Protein variants having modified immunogenicity

ABSTRACT

The present invention relates to a method of selecting a protein variant having modified immunogenicity as compared to the parent protein comprising the steps obtaining antibody binding peptide sequences, using the sequences to localise epitope sequences on the 3-dimensional structure of parent protein, defining an epitope area including amino acids situated within 5 Å from the epitope amino acids constituting the epitope sequence, changing one or more of the amino acids defining the epitope area of the parent protein by genetical engineering mutations of a DNA sequence encoding the parent protein, introducing the mutated DNA sequence into a suitable host, culturing said host and expressing the protein variant, and evaluating the immunogenicity of the protein variant using the parent protein as reference. The invention further relates to the protein variant and use thereof, as well as to a method for producing said protein variant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/957,806 filed on Sep. 21, 2001, which is a continuation ofPCT/DK01/00293 filed Apr. 30, 2001 and claims, under 35 U.S.C. 119,priority or the benefit of Danish application nos. PA 2000 00707 and PA2001 00327 filed Apr. 28, 2000 and Feb. 28, 2001, respectively, and U.S.application Nos. 60/203,345 and 60/277,817 filed May 10, 2000 and Mar.21, 2001, respectively, the contents of which are fully incorporatedherein by reference.

FIELD OF INVENTION

The present invention relates to a method of selecting a protein varianthaving modified immunogenicity as compared to the parent protein, to theprotein variant and use thereof, as well as to a method for producingsaid protein variant.

BACKGROUND OF THE INVENTION

An increasing number of proteins, including enzymes, are being producedindustrially, for use in various industries, housekeeping and medicine.Being proteins they are likely to stimulate an immunological response inman and animals, including an allergic response.

Depending on the application, individuals get sensitised to therespective allergens by inhalation, direct contact with skin and eyes,or injection. The general mechanism behind an allergic response isdivided in a sensitisation phase and a symptomatic phase. Thesensitisation phase involves a first exposure of an individual to anallergen. This event activates specific T- and B-lymphocytes, and leadsto the production of allergen specific IgE antibodies (in the presentcontext the antibodies are denoted as usual, i.e. immunoglobulin E IgEetc.). These IgE antibodies eventually facilitate allergen capturing andpresentation to T-lymphocytes at the onset of the symptomatic phase.This phase is initiated by a second exposure to the same or a resemblingantigen. The specific IgE antibodies bind to the specific IgE receptorson mast cells and basophils, among others, and capture at the same timethe allergen. The polyclonal nature of this process results in bridgingand clustering of the IgE receptors, and subsequently in the activationof mast cells and basophils. This activation triggers the release ofvarious chemical mediators involved in the early as well as late phasereactions of the symptomatic phase of allergy. Prevention of allergy insusceptible individuals is therefore a research area of greatimportance.

For certain forms of IgE-mediated allergies, a therapy exists, whichcomprises repeated administration of allergen preparations called‘allergen vaccines’ (Int. Arch. Allergy Immunol., 1999, vol. 119, pp1-5). This leads to reduction of the allergic symptoms, possibly due toa redirection of the immune response away from the allergic (Th2)pathway and towards the immunoprotective (Th1) pathway (Int. Arch.Allergy Immunol., 1999, vol. 119, pp 1-5).

Various attempts to reduce the immunogenicity of polypeptides andproteins have been conducted. It has been found that small changes in anepitope may affect the binding to an antibody. This may result in areduced importance of such an epitope, maybe converting it from a highaffinity to a low affinity epitope, or maybe even result in epitopeloss, i.e. that the epitope cannot sufficiently bind an antibody toelicit an immunogenic response.

There is a need for methods to identify epitopes on proteins and alterthese epitopes in order to modify the immunogenicity of proteins in atargeted manner. Such methods and kits for their execution can have atleast four useful purposes:

1) reduce the allergenicity of a commercial protein using proteinengineering.

2) reduce the potential of commercial proteins to cross-react withenvironmental allergens and hence cause allergic reactions in peoplesensitized to the environmental allergens (or vice versa).

3) improve the immunotherapeutic effect of allergen vaccines.

4) assist characterization of clinical allergies in order to select theappropriate treatment, including allergen vaccination.

In WO 99/53038 (Genencor Int.) as well as in prior references (Kammereret al, Clin. Exp. Allergy, 1997, vol. 27, pp 1016-1026; Sakakibara etal, J. Vet. Med. Sci., 1998; vol. 60, pp. 599-605), methods aredescribed, which identify linear T-cell epitopes among a library ofknown peptide sequences, each representing part of the primary sequenceof the protein of interest. Further, several similar techniques forlocalization of B-cell epitopes are disclosed by Walshet et al, J.Immunol. Methods, vol. 121, 1275-280, (1989), and by Schoofs et al. J.Immunol. vol. 140, 611-616, (1987). All of these methods, however, onlyleads to identification of linear epitopes, not to identification of‘structural’ or ‘discontinuous’ epitopes, which are found on the3-dimensional surface of protein molecules and which comprise aminoacids from several discrete sites of the primary sequence of theprotein. For several allergens, it has been realized that the dominantepitopes are of such discontinuous nature (Collins et al., Clin. Exp.All. 1996, vol. 26, pp. 36-42).

Slootstra et al; Molecular Diversity, 2, pp. 156-164, 1996 disclose thescreening of a semi-random library of synthetic peptides for theirbinding properties to three monoclonal antibodies by immobilizing thepeptides on polyethylene pins and binding a dilution series of eachantibody to the pins. This reference does not disclose any indication ofhow the antibody binding peptide sequences relate to any full proteinantigens or allergens.

In WO 92/10755 a method for modifying proteins to obtain lessimmunogenic variants is described. Randomly constructed proteinvariants, revealing a reduced binding of antibodies to the parent enzymeas compared to the parent enzyme itself, are selected for themeasurement in animal models in terms of allergenicity. Finally, it isassessed whether reduction in immunogenicity is due to true eliminationof an epitope or a reduction in affinity for antibodies. This methodtargets the identification of amino acids that may be part of structuralepitopes by using a complete protein for assessing antigen binding. Themajor drawbacks of this approach are the ‘trial and error’ character,which makes it a lengthy and expensive process, and the lack of generalinformation on the epitope patterns. Without this information, theresults obtained for one protein can not be applied on another protein.

WO 99/47680 (ALK-ABELLÓ) discloses the identification and modificationof B-cell epitopes by protein engineering. However, the method is basedon crystal structures of Fab-antigen complexes, and B-cell epitopes aredefined as “a section of the surface of the antigen comprising 15-25amino acid residues, which are within a distance from the atoms of theantibody enabling direct interaction” (p. 3). This publication does notshow how one selects which Fab fragment to use (e.g. to target the mostdominant allergy epitopes) or how one selects the substitutions to bemade. Further, their method cannot be used in the absence of suchcrystallographic data for antigen-antibody complexes, which are verycumbersome, sometimes impossible, to obtain—especially since one wouldneed a separate crystal structure for each epitope to be changed.

Hence, it is of interest to establish a general and efficient method toidentify structural epitopes on the 3-dimensional surface of commercialand environmental allergens.

SUMMARY OF THE INVENTION

The present invention relates to a method of selecting a protein varianthaving modified immunogenicity as compared to a parent protein,comprising the steps of:

a) obtaining antibody binding peptide sequences,

b) using the sequences to localise epitope sequences on the3-dimensional structure of parent protein,

c) defining an epitope area including amino acids situated within 5 Åfrom the epitope amino acids constituting the epitope sequence,

d) changing one or more of the amino acids defining the epitope area ofthe parent protein by genetic engineering mutations of a DNA sequenceencoding the parent protein,

e) introducing the mutated DNA sequence into a suitable host, culturingsaid host and expressing the protein variant, and

f) evaluating the immunogenicity of the protein variant using the parentprotein as reference.

A second aspect of the present invention is a protein variant havingmodified immunogenicity as compared to its parent protein. The aminoacid sequence of the protein variant differs from the amino acidsequence of the parent protein with respect to at least one epitopepattern of the parent protein, such that the immunogenicity of theprotein variant is modified as compared with the immunogenicity of theparent protein.

A further aspect of the present invention is a composition comprising aprotein variant as defined above, as well as the use of the compositionfor industrial application, such as the production of a formulation forpersonal care products (for example shampoo; soap; skin, hand and facelotions; skin, hand and face crèmes; hair dyes; toothpaste), food (forexample in the baking industry), detergents and for the production ofpharmaceuticals, e.g. vaccines.

Yet another aspect is a DNA molecule encoding a protein variant asdefined above.

Further aspects are a vector comprising a DNA molecule as describedabove as well a host cell comprising said DNA molecule.

Another aspect is a method of producing a protein variant havingmodified immunogenicity as compared to the parent protein as definedabove.

DEFINITIONS

Prior to a discussion of the detailed embodiments of the invention, adefinition of specific terms related to the main aspects of theinvention is provided.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”) DNA Cloning: A Practical Approach, Volumes Iand II/D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed.1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds(1985)); Transcription And Translation (B. D. Hames & S. J. Higgins,eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986));Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, APractical Guide To Molecular Cloning (1984).

When applied to a protein, the term “isolated” indicates that theprotein is found in a condition other than its native environment, suchas apart from blood and animal tissue. In a preferred form, the isolatedprotein is substantially free of other proteins, particularly otherproteins of animal origin. It is preferred to provide the proteins in ahighly purified form, i.e., greater than 95% pure, more preferablygreater than 99% pure. When applied to a polynucleotide molecule, theterm “isolated” indicates that the molecule is removed from its naturalgenetic milieu, and is thus free of other extraneous or unwanted codingsequences, and is in a form suitable for use within geneticallyengineered protein production systems. Such isolated molecules are thosethat are separated from their natural environment and include cDNA andgenomic clones. Isolated DNA molecules of the present invention are freeof other genes with which they are ordinarily associated, and mayinclude naturally occurring 5′ and 3′ untranslated regions such aspromoters and terminators. The identification of associated regions willbe evident to one of ordinary skill in the art (see for example, Dynanand Tijan, Nature 316: 774-78, 1985).

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”) in either singlestranded form, or a double-stranded helix. Double stranded DNA-DNA,DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary or quaternary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

A DNA “coding sequence” is a double-stranded DNA sequence, which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

An “Expression vector” is a DNA molecule, linear or circular, thatcomprises a segment encoding a polypeptide of interest operably linkedto additional segments that provide for its transcription. Suchadditional segments may include promoter and terminator sequences, andoptionally one or more origins of replication, one or more selectablemarkers, an enhancer, a polyadenylation signal, and the like. Expressionvectors are generally derived from plasmid or viral DNA, or may containelements of both.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

A “secretory signal sequence” is a DNA sequence that encodes apolypeptide (a “secretory peptide” that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

“Operably linked”, when referring to DNA segments, indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in the promoter andproceeds through the coding segment to the terminator.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced and translated into the protein encoded by the coding sequence.

“Isolated polypeptide” is a polypeptide which is essentially free ofother non-[enzyme]polypeptides, e.g., at least about 20% pure,preferably at least about 40% pure, more preferably about 60% pure, evenmore preferably about 80% pure, most preferably about 90% pure, and evenmost preferably about 95% pure, as determined by SDS-PAGE.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

A cell has been “transfected” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. A cell has been “transformed”by exogenous or heterologous DNA when the transfected DNA effects aphenotypic change. Preferably, the transforming DNA should be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

A “clone” is a population of cells derived from a single cell or commonancestor by mitosis.

“Homologous recombination” refers to the insertion of a foreign DNAsequence of a vector in a chromosome. Preferably, the vector targets aspecific chromosomal site for homologous recombination. For specifichomologous recombination, the vector will contain sufficiently longregions of homology to sequences of the chromosome to allowcomplementary binding and incorporation of the vector into thechromosome. Longer regions of homology, and greater degrees of sequencesimilarity, may increase the efficiency of homologous recombination.

Nucleic Acid Sequence

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are known in the art and include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a strain producing the polypeptide, or fromanother related organism and thus, for example, may be an allelic orspecies variant of the polypeptide encoding region of the nucleic acidsequence.

The term “isolated” nucleic acid sequence as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably about 60% pure, even more preferably about 80%pure, most preferably about 90% pure, and even most preferably about 95%pure, as determined by agarose gel electorphoresis. For example, anisolated nucleic acid sequence can be obtained by standard cloningprocedures used in genetic engineering to relocate the nucleic acidsequence from its natural location to a different site where it will bereproduced. The cloning procedures may involve excision and isolation ofa desired nucleic acid fragment comprising the nucleic acid sequenceencoding the polypeptide, insertion of the fragment into a vectormolecule, and incorporation of the recombinant vector into a host cellwhere multiple copies or clones of the nucleic acid sequence will bereplicated. The nucleic acid sequence may be of genomic, cDNA, RNA,semisynthetic, synthetic origin, or any combinations thereof.

Nucleic Acid Construct

As used herein the term “nucleic acid construct” is intended to indicateany nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNAorigin. The term “construct” is intended to indicate a nucleic acidsegment which may be single- or double-stranded, and which may be basedon a complete or partial naturally occurring nucleotide sequenceencoding a polypeptide of interest. The construct may optionally containother nucleic acid segments.

The DNA of interest may suitably be of genomic or cDNA origin, forinstance obtained by preparing a genomic or cDNA library and screeningfor DNA sequences coding for all or part of the polypeptide byhybridization using synthetic oligonucleotide probes in accordance withstandard techniques (cf. Sambrook et al., supra).

The nucleic acid construct may also be prepared synthetically byestablished standard methods, e.g. the phosphoamidite method describedby Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, orthe method described by Matthes et al., EMBO Journal 3 (1984), 801-805.According to the phosphoamidite method, oligonucleotides aresynthesized, e.g. in an automatic DNA synthesizer, purified, annealed,ligated and cloned in suitable vectors.

Furthermore, the nucleic acid construct may be of mixed synthetic andgenomic, mixed synthetic and cDNA or mixed genomic and cDNA originprepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate), the fragments corresponding to various parts of the entirenucleic acid construct, in accordance with standard techniques.

The nucleic acid construct may also be prepared by polymerase chainreaction using specific primers, for instance as described in U.S. Pat.No. 4,683,202 or Saiki et al., Science 239 (1988), 487-491.

The term nucleic acid construct may be synonymous with the termexpression cassette when the nucleic acid construct contains all thecontrol sequences required for expression of a coding sequence of thepresent invention. The term “coding sequence” as defined herein is asequence which is transcribed into mRNA and translated into apolypeptide of the present invention when placed under the control ofthe above mentioned control sequences. The boundaries of the codingsequence are generally determined by a translation start codon ATG atthe 5′-terminus and a translation stop codon at the 3′-terminus. Acoding sequence can include, but is not limited to, DNA, cDNA, andrecombinant nucleic acid sequences.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for expression of the codingsequence of the nucleic acid sequence. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader, apolyadenylation sequence, a propeptide sequence, a promoter, a signalsequence, and a transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acidsequence encoding a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcription andtranslation control sequences which mediate the expression of thepolypeptide. The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice and may be obtainedfrom genes encoding extracellular or intracellular polypeptides eitherhomologous or heterologous to the host cell.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′ terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

The control sequence may also be a signal peptide coding region, whichcodes for an amino acid sequence linked to the amino terminus of thepolypeptide which can direct the expressed polypeptide into the cell'ssecretory pathway of the host cell. The 5′ end of the coding sequence ofthe nucleic acid sequence may inherently contain a signal peptide codingregion naturally linked in translation reading frame with the segment ofthe coding region which encodes the secreted polypeptide.

Alternatively, the 5′ end of the coding sequence may contain a signalpeptide coding region which is foreign to that portion of the codingsequence which encodes the secreted polypeptide. A foreign signalpeptide coding region may be required where the coding sequence does notnormally contain a signal peptide coding region. Alternatively, theforeign signal peptide coding region may simply replace the naturalsignal peptide coding region in order to obtain enhanced secretionrelative to the natural signal peptide coding region normally associatedwith the coding sequence. The signal peptide coding region may beobtained from a glucoamylase or an amylase gene from an Aspergillusspecies, a lipase or proteinase gene from a Rhizomucor species, the genefor the alpha-factor from Saccharomyces cerevisiae, an amylase or aprotease gene from a Bacillus species, or the calf preprochymosin gene.However, any signal peptide coding region capable of directing theexpressed polypeptide into the secretory pathway of a host cell ofchoice may be used in the present invention.

The control sequence may also be a propeptide coding region, which codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to mature active polypeptide bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from theBacillus subtilis alkaline protease gene (aprE), the Bacillus subtilisneutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factorgene, or the Myceliophthora thermophilum laccase gene (WO 95/33836).

The nucleic acid constructs of the present invention may also compriseone or more nucleic acid sequences which encode one or more factors thatare advantageous in the expression of the polypeptide, e.g., anactivator (e.g., a trans-acting factor), a chaperone, and a processingprotease. Any factor that is functional in the host cell of choice maybe used in the present invention. The nucleic acids encoding one or moreof these factors are not necessarily in tandem with the nucleic acidsequence encoding the polypeptide.

An activator is a protein which activates transcription of a nucleicacid sequence encoding a polypeptide (Kudla et al., 1990, EMBO Journal9:1355-1364; Jarai and Buxton, 1994, Current Genetics 26:2238-244;Verdier, 1990, Yeast 6:271-297). The nucleic acid sequence encoding anactivator may be obtained from the genes encoding Bacillusstearothermophilus NprA (nprA), Saccharomyces cerevisiae heme activatorprotein 1 (hap1), Saccharomyces cerevisiae galactose metabolizingprotein 4 (gal4), and Aspergillus nidulans ammonia regulation protein(areA). For further examples, see Verdier, 1990, supra and MacKenzie etal., 1993, Journal of General Microbiology 139:2295-2307.

A chaperone is a protein which assists another polypeptide in foldingproperly (Hartl et al., 1994, TIBS 19:20-25; Bergeron et al., 1994, TIBS19:124-128; Demolder et al., 1994, Journal of Biotechnology 32:179-189;Craig, 1993, Science 260:1902-1903; Gething and Sambrook, 1992, Nature355:33-45; Puig and Gilbert, 1994, Journal of Biological Chemistry269:7764-7771; Wang and Tsou, 1993, The FASEB Journal 7:1515-11157;Robinson et al., 1994, Bio/Technology 1:381-384). The nucleic acidsequence encoding a chaperone may be obtained from the genes encodingBacillus subtilis GroE proteins, Aspergillus oryzae protein disulphideisomerase, Saccharomyces cerevisiae calnexin, Saccharomyces cerevisiaeBiP/GRP78, and Saccharomyces cerevisiae Hsp70. For further examples, seeGething and Sambrook, 1992, supra, and Hartl et al., 1994, supra.

A processing protease is a protease that cleaves a propeptide togenerate a mature biochemically active polypeptide (Enderlin andOgrydziak, 1994, Yeast 10:67-79; Fuller et al., 1989, Proceedings of theNational Academy of Sciences USA 86:1434-1438; Julius et al., 1984, Cell37:1075-1089; Julius et al., 1983, Cell 32:839-852). The nucleic acidsequence encoding a processing protease may be obtained from the genesencoding Aspergillus niger Kex2, Saccharomyces cerevisiaedipeptidylaminopeptidase, Saccharomyces cerevisiae Kex2, and Yarrowialipolytica dibasic processing endoprotease (xpr6).

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems would include thelac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1system may be used. In filamentous fungi, the TAKA alpha-amylasepromoter, Aspergillus niger glucoamylase promoter, and the Aspergillusoryzae glucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beplaced in tandem with the regulatory sequence.

Promoters

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, the Streptomyces coelicolor agarase gene (dagA), the Bacillussubtilis levansucrase gene (sacB), the Bacillus subtilis alkalineprotease gene, the Bacillus licheniformis alpha-amylase gene (amyL), theBacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillusamyloliquefaciens alpha-amylase gene (amyQ), the Bacillusamyloliquefaciens BAN amylase gene, the Bacillus licheniformispenicillinase gene (penP), the Bacillus subtilis xylA and xylB genes,and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75:3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80:21-25), or the Bacillus pumilusxylosidase gene, or by the phage Lambda PR or PL promoters or the E.coli lac, trp or tac promoters. Further promoters are described in“Useful proteins from recombinant bacteria” in Scientific American,1980, 242:74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes encoding Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, Fusarium oxysporum trypsin-like protease (as described inU.S. Pat. No. 4,288,627, which is incorporated herein by reference), andhybrids thereof. Particularly preferred promoters for use in filamentousfungal host cells are the TAKA amylase, NA2-tpi (a hybrid of thepromoters from the genes encoding Aspergillus niger neutralalpha-amylase and Aspergillus oryzae triose phosphate isomerase), andglaA promoters. Further suitable promoters for use in filamentous fungushost cells are the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985),2093-2099) or the tpiA promoter.

Examples of suitable promoters for use in yeast host cells includepromoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem.255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1(1982), 419-434) or alcohol dehydrogenase genes (Young et al., inGenetic Engineering of Microorganisms for Chemicals (Hollaender et al,eds.), Plenum Press, New York, 1982), or the TPI1 (U.S. Pat. No.4,599,311) or ADH2-4-c (Russell et al., Nature 304 (1983), 652-654)promoters.

Further useful promoters are obtained from the Saccharomyces cerevisiaeenolase (ENO-1) gene, the Saccharomyces cerevisiae galactokinase gene(GAL1), the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP),and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Otheruseful promoters for yeast host cells are described by Romanos et al.,1992, Yeast 8:423-488. In a mammalian host cell, useful promotersinclude viral promoters such as those from Simian Virus 40 (SV40), Roussarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).

Examples of suitable promoters for directing the transcription of theDNA encoding the polypeptide of the invention in mammalian cells are theSV40 promoter (Subramani et al., Mol. Cell. Biol. 1 (1981), 854-864),the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222(1983), 809-814) or the adenovirus 2 major late promoter.

An example of a suitable promoter for use in insect cells is thepolyhedrin promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al., FEBSLett. 311, (1992) 7-11), the P10 promoter (J. M. Vlak et al., J. Gen.Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosisvirus basic protein promoter (EP 397 485), the baculovirus immediateearly gene 1 promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No.5,162,222), or the baculovirus 39K delayed-early gene promoter (U.S.Pat. No. 5,155,037; U.S. Pat. No. 5,162,222).

Terminators

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes encoding Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase,Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-likeprotease. for fungal hosts) the TPI1 (Alber and Kawasaki, op. cit.) orADH3 (McKnight et al., op. cit.) terminators.

Preferred terminators for yeast host cells are obtained from the genesencoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), or Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

Polyadenylation Signals

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes encoding Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, and Aspergillus niger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15:5983-5990.

Polyadenylation sequences are well known in the art for mammalian hostcells such as SV40 or the adenovirus 5 Elb region.

Signal Sequences

An effective signal peptide coding region for bacterial host cells isthe signal peptide coding region obtained from the maltogenic amylasegene from Bacillus NCIB 11837, the Bacillus stearothermophilusalpha-amylase gene, the Bacillus licheniformis subtilisin gene, theBacillus licheniformis beta-lactamase gene, the Bacillusstearothermophilus neutral proteases genes (nprT, nprS, nprM), and theBacillus subtilis PrsA gene. Further signal peptides are described bySimonen and Palva, 1993, Microbiological Reviews 57:109-137.

An effective signal peptide coding region for filamentous fungal hostcells is the signal peptide coding region obtained from Aspergillusoryzae TAKA amylase gene, Aspergillus niger neutral amylase gene, theRhizomucor miehei aspartic proteinase gene, the Humicola lanuginosacellulase or lipase gene, or the Rhizomucor miehei lipase or proteasegene, Aspergillus sp. amylase or glucoamylase, a gene encoding aRhizomucor miehei lipase or protease. The signal peptide is preferablyderived from a gene encoding A. oryzae TAKA amylase, A. niger neutralalpha-amylase, A. niger acid-stable amylase, or A. niger glucoamylase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

For secretion from yeast cells, the secretory signal sequence may encodeany signal peptide which ensures efficient direction of the expressedpolypeptide into the secretory pathway of the cell. The signal peptidemay be naturally occurring signal peptide, or a functional part thereof,or it may be a synthetic peptide. Suitable signal peptides have beenfound to be the a-factor signal peptide (cf. U.S. Pat. No. 4,870,008),the signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et al.,Nature 289, 1981, pp. 643-646), a modified carboxypeptidase signalpeptide (cf. L. A. Valls et al., Cell 48, 1987, pp. 887-897), the yeastBAR1 signal peptide (cf. WO 87/02670), or the yeast aspartic protease 3(YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp.127-137).

For efficient secretion in yeast, a sequence encoding a leader peptidemay also be inserted downstream of the signal sequence and upstream ofthe DNA sequence encoding the polypeptide. The function of the leaderpeptide is to allow the expressed polypeptide to be directed from theendoplasmic reticulum to the Golgi apparatus and further to a secretoryvesicle for secretion into the culture medium (i.e. exportation of thepolypeptide across the cell wall or at least through the cellularmembrane into the periplasmic space of the yeast cell). The leaderpeptide may be the yeast a-factor leader (the use of which is describedin e.g. U.S. Pat. No. 4,546,082, EP 16 201, EP 123 294, EP 123 544 andEP 163 529). Alternatively, the leader peptide may be a synthetic leaderpeptide, which is to say a leader peptide not found in nature. Syntheticleader peptides may, for instance, be constructed as described in WO89/02463 or WO 92/11378.

For use in insect cells, the signal peptide may conveniently be derivedfrom an insect gene (cf. WO 90/05783), such as the lepidopteran Manducasexta adipokinetic hormone precursor signal peptide (cf. U.S. Pat. No.5,023,328).

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the nucleic acid sequence of the present invention may beexpressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression, and possiblysecretion.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids. The vector may be anautonomously replicating vector, i.e., a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome. The vector may contain anymeans for assuring self-replication. Alternatively, the vector may beone which, when introduced into the host cell, is integrated into thegenome and replicated together with the chromosome(s) into which it hasbeen integrated. The vector system may be a single vector or plasmid ortwo or more vectors or plasmids which together contain the total DNA tobe introduced into the genome of the host cell, or a transposon.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol, tetracycline, neomycin, hygromycin or methotrexateresistance. A frequently used mammalian marker is the dihydrofolatereductase gene (DHFR). Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A selectable marker for use in afilamentous fungal host cell may be selected from the group including,but not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), and glufosinate resistance markers, aswell as equivalents from other species. Preferred for use in anAspergillus cell are the amdS and pyrG markers of Aspergillus nidulansor Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus.Furthermore, selection may be accomplished by co-transformation, e.g.,as described in WO 91/17243, where the selectable marker is on aseparate vector.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell genomeor autonomous replication of the vector in the cell independent of thegenome of the cell.

The vectors of the present invention may be integrated into the hostcell genome when introduced into a host cell. For integration, thevector may rely on the nucleic acid sequence encoding the polypeptide orany other element of the vector for stable integration of the vectorinto the genome by homologous or nonhomologous recombination.Alternatively, the vector may contain additional nucleic acid sequencesfor directing integration by homologous recombination into the genome ofthe host cell. The additional nucleic acid sequences enable the vectorto be integrated into the host cell genome at a precise location(s) inthe chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 1,500 base pairs,preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500base pairs, which are highly homologous with the corresponding targetsequence to enhance the probability of homologous recombination. Theintegrational elements may be any sequence that is homologous with thetarget sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleic acidsequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination. These nucleicacid sequences may be any sequence that is homologous with a targetsequence in the genome of the host cell, and, furthermore, may benon-encoding or encoding sequences.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, pACYC184,pUB110, pE194, pTA1060, and pAMβ1. Examples of origin of replicationsfor use in a yeast host cell are the 2 micron origin of replication, thecombination of CEN6 and ARS4, and the combination of CEN3 and ARS1. Theorigin of replication may be one having a mutation which makes itsfunctioning temperature-sensitive in the host cell (see, e.g., Ehrlich,1978, Proceedings of the National Academy of Sciences USA 75:1433).

More than one copy of a nucleic acid sequence encoding a polypeptide ofthe present invention may be inserted into the host cell to amplifyexpression of the nucleic acid sequence. Stable amplification of thenucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome using methodswell known in the art and selecting for transformants.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga nucleic acid sequence of the invention, which are advantageously usedin the recombinant production of the polypeptides. The term “host cell”encompasses any progeny of a parent cell which is not identical to theparent cell due to mutations that occur during replication.

The cell is preferably transformed with a vector comprising a nucleicacid sequence of the invention followed by integration of the vectorinto the host chromosome. “Transformation” means introducing a vectorcomprising a nucleic acid sequence of the present invention into a hostcell so that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector. Integration is generallyconsidered to be an advantage as the nucleic acid sequence is morelikely to be stably maintained in the cell. Integration of the vectorinto the host chromosome may occur by homologous or non-homologousrecombination as described above.

The choice of a host cell will to a large extent depend upon the geneencoding the polypeptide and its source. The host cell may be aunicellular microorganism, e.g., a prokaryote, or a non-unicellularmicroorganism, e.g., a eukaryote. Useful unicellular cells are bacterialcells such as gram positive bacteria including, but not limited to, aBacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans orStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred embodiment, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus orBacillus subtilis cell. The transformation of a bacterial host cell may,for instance, be effected by protoplast transformation (see, e.g., Changand Cohen, 1979, Molecular General Genetics 168:111-115), by usingcompetent cells (see, e.g., Young and Spizizin, 1961, Journal ofBacteriology 81:823-829, or Dubnar and Davidoff-Abelson, 1971, Journalof Molecular Biology 56:209-221), by electroporation (see, e.g.,Shigekawa and Dower, 1988, Biotechniques 6:742-751), or by conjugation(see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology169:5771-5278).

The host cell may be a eukaryote, such as a mammalian cell, an insectcell, a plant cell or a fungal cell.

Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney (BHK) cells, COS cells, or any number ofother immortalized cell lines available, e.g., from the American TypeCulture Collection.

Examples of suitable mammalian cell lines are the COS (ATCC CRL 1650 and1651), BHK (ATCC CRL 1632, 10314 and 1573, ATCC CCL 10), CHL (ATCCCCL39) or CHO (ATCC CCL 61) cell lines. Methods of transfectingmammalian cells and expressing DNA sequences introduced in the cells aredescribed in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621;Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327-341; Loyter etal., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981),603, Ausubel et al., Current Protocols in Molecular Biology, John Wileyand Sons, Inc., N.Y., 1987, Hawley-Nelson et al., Focus 15 (1993), 73;Ciccarone et al., Focus 15 (1993), 80; Graham and van der Eb, Virology52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

In a preferred embodiment, the host cell is a fungal cell. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra). Representative groupsof Ascomycota include, e.g., Neurospora, Eupenicillium (=Penicillium),Emericella (=Aspergillus), Eurotium (=Aspergillus), and the true yeastslisted above. Examples of Basidiomycota include mushrooms, rusts, andsmuts. Representative groups of Chytridiomycota include, e.g.,Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi.Representative groups of Oomycota include, e.g., Saprolegniomycetousaquatic fungi (water molds) such as Achlya. Examples of mitosporic fungiinclude Aspergillus, Penicillium, Candida, and Alternaria.Representative groups of Zygomycota include, e.g., Rhizopus and Mucor.

In a preferred embodiment, the fungal host cell is a yeast cell. “Yeast”as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). The ascosporogenous yeasts are divided into thefamilies Spermophthoraceae and Saccharomycetaceae. The latter iscomprised of four subfamilies, Schizosaccharomycoideae (e.g., genusSchizosaccharomyces), Nadsonioideae, Lipomycoideae, andSaccharomycoideae (e.g., genera Pichia, Kluyveromyces andSaccharomyces). The basidiosporogenous yeasts include the generaLeucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, andFilobasidiella. Yeast belonging to the Fungi Imperfecti are divided intotwo families, Sporobolomycetaceae (e.g., genera Sorobolomyces andBullera) and Cryptococcaceae (e.g., genus Candida). Since theclassification of yeast may change in the future, for the purposes ofthis invention, yeast shall be defined as described in Biology andActivities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980. The biologyof yeast and manipulation of yeast genetics are well known in the art(see, e.g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B.J., and Stopani, A. O. M., editors, 2nd edition, 1987; The Yeasts, Rose,A. H., and Harrison, J. S., editors, 2nd edition, 1987; and TheMolecular Biology of the Yeast Saccharomyces, Strathern et al., editors,1981).

The yeast host cell may be selected from a cell of a species of Candida,Kluyveromyces, Saccharomyces, Schizosaccharomyces, Candida, Pichia,Hansenula, or Yarrowia. In a preferred embodiment, the yeast host cellis a Saccharomyces carlsbergensis, Saccharomyces cerevisiae,Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyceskluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. Otheruseful yeast host cells are a Kluyveromyces lactis, Kluyveromycesfragilis, Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica,Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose, Pichiaguillermondii and Pichia methanolio cell (cf. Gleeson et al., J. Gen.Microbiol. 132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279 and U.S.Pat. No. 4,879,231).

In a preferred embodiment, the fungal host cell is a filamentous fungalcell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are characterized by a vegetativemycelium composed of chitin, cellulose, glucan, chitosan, mannan, andother complex polysaccharides. Vegetative growth is by hyphal elongationand carbon catabolism is obligately aerobic. In contrast, vegetativegrowth by yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative. In a morepreferred embodiment, the filamentous fungal host cell is a cell of aspecies of, but not limited to, Acremonium, Aspergillus, Fusarium,Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia,Tolypocladium, and Trichoderma or a teleomorph or synonym thereof. In aneven more preferred embodiment, the filamentous fungal host cell is anAspergillus cell. In another even more preferred embodiment, thefilamentous fungal host cell is an Acremonium cell. In another even morepreferred embodiment, the filamentous fungal host cell is a Fusariumcell. In another even more preferred embodiment, the filamentous fungalhost cell is a Humicola cell. In another even more preferred embodiment,the filamentous fungal host cell is a Mucor cell. In another even morepreferred embodiment, the filamentous fungal host cell is aMyceliophthora cell. In another even more preferred embodiment, thefilamentous fungal host cell is a Neurospora cell. In another even morepreferred embodiment, the filamentous fungal host cell is a Penicilliumcell. In another even more preferred embodiment, the filamentous fungalhost cell is a Thielavia cell. In another even more preferredembodiment, the filamentous fungal host cell is a Tolypocladium cell. Inanother even more preferred embodiment, the filamentous fungal host cellis a Trichoderma cell. In a most preferred embodiment, the filamentousfungal host cell is an Aspergillus awamori, Aspergillus foetidus,Aspergillus japonicus, Aspergillus niger, Aspergillus nidulans orAspergillus oryzae cell. In another most preferred embodiment, thefilamentous fungal host cell is a Fusarium cell of the section Discolor(also known as the section Fusarium). For example, the filamentousfungal parent cell may be a Fusarium bactridioides, Fusarium cerealis,Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusariumreticulaturn, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sulphureum, or Fusarium trichothecioides cell. Inanother preferred embodiment, the filamentous fungal parent cell is aFusarium strain of the section Elegans, e.g., Fusarium oxysporum. Inanother most preferred embodiment, the filamentous fungal host cell is aHumicola insolens or Humicola lanuginosa cell. In another most preferredembodiment, the filamentous fungal host cell is a Mucor miehei cell. Inanother most preferred embodiment, the filamentous fungal host cell is aMyceliophthora thermophilum cell. In another most preferred embodiment,the filamentous fungal host cell is a Neurospora crassa cell. In anothermost preferred embodiment, the filamentous fungal host cell is aPenicillium purpurogenum cell. In another most preferred embodiment, thefilamentous fungal host cell is a Thielavia terrestris cell or anAcremonium chtysogenum cell. In another most preferred embodiment, theTrichoderma cell is a Trichoderma harzianum, Trichoderma koningii,Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viridecell. The use of Aspergillus spp. for the expression of proteins isdescribed in, e.g., EP 272 277, EP 230 023.

Transformation

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81:1470-1474. A suitable method of transforming Fusarium species isdescribed by Malardier et al., 1989, Gene 78:147-156 or in copendingU.S. Ser. No. 08/269,449. Examples of other fungal cells are cells offilamentous fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp.or Trichoderma spp., in particular strains of A. oryzae, A. nidulans orA. niger. The use of Aspergillus spp. for the expression of proteins isdescribed in, e.g., EP 272 277, EP 230 023. The transformation of F.oxysporum may, for instance, be carried out as described by Malardier etal., 1989, Gene 78: 147-156.

Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153:163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75:1920. Mammalian cells may betransformed by direct uptake using the calcium phosphate precipitationmethod of Graham and Van der Eb (1978, Virology 52:546).

Transformation of insect cells and production of heterologouspolypeptides therein may be performed as described in U.S. Pat. No.4,745,051; U.S. Pat. No. 4,775,624; U.S. Pat. No. 4,879,236; U.S. Pat.No. 5,155,037; U.S. Pat. No. 5,162,222; EP 397,485) all of which areincorporated herein by reference. The insect cell line used as the hostmay suitably be a Lepidoptera cell line, such as Spodoptera frugiperdacells or Trichoplusia ni cells (cf. U.S. Pat. No. 5,077,214). Cultureconditions may suitably be as described in, for instance, WO 89/01029 orWO 89/01028, or any of the aforementioned references.

Methods of Production

The transformed or transfected host cells described above are culturedin a suitable nutrient medium under conditions permitting the productionof the desired molecules, after which these are recovered from thecells, or the culture broth.

The medium used to culture the cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection). The mediaare prepared using procedures known in the art (see, e.g., referencesfor bacteria and yeast; Bennett, J. W. and LaSure, L., editors, MoreGene Manipulations in Fungi, Academic Press, CA, 1991).

If the molecules are secreted into the nutrient medium, they can berecovered directly from the medium. If they are not secreted, they canbe recovered from cell lysates. The molecules are recovered from theculture medium by conventional procedures including separating the hostcells from the medium by centrifugation or filtration, precipitating theproteinaceous components of the supernatant or filtrate by means of asalt, e.g. ammonium sulphate, purification by a variety ofchromatographic procedures, e.g. ion exchange chromatography,gelfiltration chromatography, affinity chromatography, or the like,dependent on the type of molecule in question.

The molecules of interest may be detected using methods known in the artthat are specific for the molecules. These detection methods may includeuse of specific antibodies, formation of a product, or disappearance ofa substrate. For example, an enzyme assay may be used to determine theactivity of the molecule. Procedures for determining various kinds ofactivity are known in the art.

The molecules of the present invention may be purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J-C Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

The term “immunological response”, used in connection with the presentinvention, is the response of an organism to a compound, which involvesthe immune system according to any of the four standard reactions (TypeI, II, III and IV according to Coombs & Gell).

Correspondingly, the “immunogenicity” of a compound used in connectionwith the present invention refers to the ability of this compound toinduce an ‘immunological response’ in animals including man.

The term “allergic response”, used in connection with the presentinvention, is the response of an organism to a compound, which involvesIgE mediated responses (Type I reaction according to Coombs & Gell). Itis to be understood that sensibilization (i.e. development ofcompound-specific IgE antibodies) upon exposure to the compound isincluded in the definition of “allergic response”.

Correspondingly, the “allergenicity” of a compound used in connectionwith the present invention refers to the ability of this compound toinduce an ‘allergic response’ in animals including man.

The term “parent protein” refer to the polypeptide to be modified bycreating a library of diversified mutants. The “parent protein” may be anaturally occurring (or wild-type) polypeptide or it may be a variantthereof prepared by any suitable means. For instance, the “parentprotein” may be a variant of a naturally occurring polypeptide which hasbeen modified by substitution, deletion or truncation of one or moreamino acid residues or by addition or insertion of one or more aminoacid residues to the amino acid sequence of a naturally-occurringpolypeptide.

The term “enzyme variants” or “protein variants” refer to a polypeptideof the invention comprising one or more substitutions of the specifiedamino acid residues. The total number of such substitutions is typicallynot more than 10, e.g. one, two, three, four, five or six of saidsubstitutions. In addition, the enzyme variant or protein variant of theinvention may optionally include other modifications of the parentenzyme, typically not more than 10, e.g. not more than 5 suchmodifications. The variant generally has a homology with the parentenzyme of at least 80%, e.g. at least 85%, typically at least 90% or atleast 95%.

The term “randomized library” of protein variants refers to a librarywith at least partially randomized composition of the members, e.g.protein variants.

An “epitope” is a set of amino acids on a protein that are involved inan immunological response, such as antibody binding or T-cellactivation. One particularly useful method of identifying epitopesinvolved in antibody binding is to screen a library of peptide-phagemembrane protein fusions and selecting those that bind to relevantantigen-specific antibodies, sequencing the randomized part of thefusion gene, aligning the sequences involved in binding, definingconsensus sequences based on these alignments, and mapping theseconsensus sequences on the surface or the sequence and/or structure ofthe antigen, to identify epitopes involved in antibody binding.

By the term “epitope pattern” is meant such a consensus sequence ofantibody binding peptides. An example is the epitope pattern A R R<R.The sign “<” in this notation indicates that the aligned antibodybinding peptides included a non-consensus amino acid between the secondand the third arginine.

An “epitope area” is defined as the amino acids situated close to theepitope sequence amino acids. Preferably, the amino acids of an epitopearea are located <5 Å from the epitope sequence. Hence, an epitope areaalso includes the corresponding epitope sequence itself. Modificationsof amino acids of the ‘epitope area’ can possibly affect the immunogenicfunction of the corresponding epitope.

By the term “epitope sequence” is meant the amino acid residues of aparent protein, which have been identified to belong to an epitope bythe methods of the present invention (an example of an epitope sequenceis E271 Q12 I8 in Savinase).

The term ‘antibody binding peptide’ denotes a peptide that bind withsufficiently high affinity to antibodies. Identification of ‘antibodybinding peptides’ and their sequences constitute the first step of themethod of this invention.

“Anchor amino acids” are the individual amino acids of an epitopepattern.

“Hot spot amino acids” are amino acids of parent protein, which areparticularly likely to result in modified immunogenecity if they aremutated. Amino acids, which appear in three or more epitope sequences orwhich correspond to anchor amino acids are hot spot amino acids.

“Environmental allergens” are protein allergens that are presentnaturally. They include pollen, dust mite allergens, pet allergens, foodallergens, venoms, etc.

“Commercial allergens” are protein allergens that are being brought tothe market commercially. They include enzymes, pharmaceutical proteins,antimicrobial peptides, as well as allergens of transgenic plants.

The “donor protein” is the protein that was used to raise antibodiesused to identify antibody binding sequences, hence the donor proteinprovides the information that leads to the epitope patterns.

The “acceptor protein” is the protein, whose structure is used to fitthe identified epitope patterns and/or to fit the antibody bindingsequences. Hence the acceptor protein is also the parent protein.

An “autoepitope” is one that has been identified using antibodies raisedagainst the parent protein, i.e. the acceptor and the donor proteins areidentical.

A “heteroepitope” is one that has been identified with distinct donorand acceptor proteins.

The term “functionality” of protein variants refers to e.g. enzymaticactivity; binding to a ligand or receptor; stimulation of a cellularresponse (e.g. ³H-thymidine incorporation as response to a mitogenicfactor); or anti-microbial activity.

By the term “specific polyclonal antibodies” is meant polyclonalantibodies isolated according to their specificity for a certainantigen, e.g. the protein backbone.

By the term “monospecific antibodies” is meant polyclonal antibodiesisolated according to their specificity for a certain epitope. Suchmonospecific antibodies will bind to the same epitope, but withdifferent affinity, as they are produced by a number of antibodyproducing cells recognizing overlapping but not necessarily identicalepitopes.

The term “randomized library” of protein variants refers to a librarywith at least partially randomized composition of the members, e.g.protein variants.

‘Spiked mutagenesis’ is a form of site-directed mutagenesis, in whichthe primers used have been synthesized using mixtures ofoligonucleotides at one or more positions.

By the term “a protein variant having modified immunogenicity ascompared to the parent protein” is meant a protein variant which differsfrom the parent protein in one or more amino acids whereby theimmunogenicity of the variant is modified. The modification ofimmunogenicity may be confirmed by testing the ability of the proteinvariant to elicit an IgE/IgG response.

In the present context the term “protein” is intended to coveroligopeptides, polypeptides as well as proteins as such.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of selecting a protein varianthaving modified immunogenicity as compared to a parent protein,comprising the steps of:

a) obtaining antibody binding peptide sequences,

b) using the sequences to localise epitope sequences on the3-dimensional structure of parent protein,

c) defining an epitope area including amino acids situated within 5 Åfrom the epitope amino acids constituting the epitope sequence,

d) changing one or more of the amino acids defining the epitope area ofthe parent protein by genetic engineering mutations of a DNA sequenceencoding the parent protein,

e) introducing the mutated DNA sequence into a suitable host, culturingsaid host and expressing the protein variant, and

f) evaluating the immunogenicity of the protein variant using the parentprotein as reference.

A) How to Find Antibody Binding Peptide Sequences and Epitope Patterns

A first step of the method is to identify peptide sequences, which bindspecifically to antibodies.

Antibody binding peptide sequences can be found by testing a set ofknown peptide sequences for binding to antibodies raised against thedonor protein. These sequences are typically selected, such that eachrepresents a segment of the donor protein sequence (Mol. Immunol., 1992,vol. 29, pp. 1383-1389; Am. J. Resp. Cell. Mol. Biol. 2000, vol. 22, pp.344-351). Also, randomized synthetic peptide libraries can be used tofind antibody binding sequences (Slootstra et al; Molecular Diversity,1996, vol. 2, pp. 156-164).

In a preferred method, the identification of antibody binding sequencesmay be achieved by screening of a display package library, preferably aphage display library. The principle behind phage display is that aheterologous DNA sequence can be inserted in the gene coding for a coatprotein of the phage (WO 92/15679). The phage will make and display thehybrid protein on its surface where it can interact with specific targetagents. Such target agent may be antigen-specific antibodies. It istherefore possible to select specific phages that displayantibody-binding peptide sequences. The displayed peptides can be ofpredetermined lengths, for example 9 amino acids long, with randomizedsequences, resulting in a random peptide display package library. Thus,by screening for antibody binding, one can isolate the peptide sequencesthat have sufficiently high affinity for the particular antibody used.The peptides of the hybrid proteins of the specific phages which bindprotein-specific antibodies characterize epitopes that are recognized bythe immune system.

The antibodies used for reacting with the display package are preferablyIgE antibodies to ensure that the epitopes identified are IgE epitopes,i.e. epitopes inducing and binding IgE. In a preferred embodiment theantibodies are polyclonal antibodies, optionally monospecificantibodies.

For the purpose of the present invention polyclonal antibodies arepreferred in order to obtain a broader knowledge about the epitopes of aprotein.

It is of great importance that the amino acid sequence of the peptidespresented by the display packages is long enough to represent asignificant part of the epitope to be identified. In a preferredembodiment of the invention the peptides of the peptide display packagelibrary are oligopeptides having from 5 to 25 amino acids, preferably atleast 8 amino acids, such as 9 amino acids. For a given length ofpeptide sequences (n), the theoretical number of different possiblesequences can be calculated as 20^(n). The diversity of the packagelibrary used must be large enough to provide a suitable representationof the theoretical number of different sequences. In a phage-displaylibrary, each phage has one specific sequence of a determined length.Hence an average phage display library can express 10⁸-10¹² differentrandom sequences, and is therefore well-suited to represent thetheoretical number of different sequences.

The antibody binding peptide sequences can be further analysed byconsensus alignment e.g. by the methods described by Feng and Doolittle,Meth. Enzymol., 1996, vol. 266, pp. 368-382; Feng and Doolittle, J. Mol.Evol., 1987, vol. 25, pp. 351-360; and Taylor, Meth. Enzymol., 1996,vol. 266, pp. 343-367.

This leads to identification of epitope patterns, which can assist thecomparison of the linear information obtained from the antibody bindingpeptide sequences to the 3-o dimensional structure of the acceptorprotein in order to identify epitope sequences at the surface of theacceptor protein.

B) How to Identify Epitope Sequences and Epitope Areas.

Given a number of antibody binding peptide sequences and possibly thecorresponding epitope patterns, one need the 3-dimensional structurecoordinates of an acceptor protein to find the epitope sequences on itssurface.

These coordinates can be found in databases (NCBI:http://www.ncbi.nlm.nih.gov/), determined experimentally usingconventional methods (Ducruix and Giege: Crystallization of NucleicAcids and Proteins, IRL PRess, Oxford, 1992, ISBN 0-19-963245-6), orthey can be deduced from the coordinates of a homologous protein.Typical actions required for the construction of a model structure are:alignment of homologous sequences for which 3-dimensional structuresexist, definition of Structurally Conserved Regions (SCRs), assignmentof coordinates to SCRs, search for structural fragments/loops instructure databases to replace Variable Regions, assignment ofcoordinates to these regions, and structural refinement by energyminimization. Regions containing large inserts (>3 residues) relative tothe known 3-dimensional structures are known to be quite difficult tomodel, and structural predictions must be considered with care.

Using the coordinates and the several methods of mapping the linearinformation on the 3-dimensional surface are possible, as described inthe examples below.

One can match each amino acid residue of the antibody binding peptide toan identical or homologous amino acid on the 3-D surface of the acceptorprotein, such that amino acids that are adjacent in the primary sequenceare close on the surface of the acceptor protein, with close being <5 Å,preferably <3 Å between any two atoms of the two amino acids.

Alternatively, one can define a geometric body (e.g. an ellipsoid, asphere, or a box) of a size that matches a possible binding interfacebetween antibody and antigen and look for a positioning of this bodywhere it will contain most of or all the anchor amino acids.

Also, one can use the epitope patterns to facilitate identification ofepitope sequences. This can be done, by first matching the anchor aminoacids on the 3-D structure and subsequently looking for other elementsof the antibody binding peptide sequences, which provide additionalmatches. If there are many residues to be matched, it is only necessarythat a suitable number can be found on the 3-D structure. For example ifan epitope pattern comprises 4, 5, 6, or 7 amino acids, it is onlynecessary that 3 matches surface elements of the acceptor protein.

In all cases, it is desirable that amino acids of the epitope sequenceare surface exposed (as described below in Examples).

It is known, that amino acids that surround binding sequences can affectbinding of a ligand without participating actively in the bindingprocess. Based on this knowledge, areas covered by amino acids withpotential steric effects on the epitope-antibody interaction, weredefined around the identified epitope sequences. These areas are called‘epitope areas’. Practically, all amino acids situated within 5 Å fromthe amino acids defining the epitope sequence were included. Preferably,the epitope area equals the epitope sequence. The accessibilitycriterium was not used as hidden amino acids of an epitope area also canhave an effect on the adjacent amino acids of the epitope sequence.

C) How to Use the Epitope Information.

There are at least four ways to utilize the information about epitopesequences, which has been derived by the methods of this invention:

1) reduce the allergenicity of a commercial protein using proteinengineering.

2) reduce the potential of commercial proteins to cross-react withenvironmental allergens and hence cause allergic reactions in peoplesensitized to the environmental allergens (or vice versa).

3) improve the immunotherapeutic effect of allergen vaccines.

4) assist characterization of clinical allergies in order to select theappropriate allergen vaccine.

Protein Engineering to Reduce the Allergenicity, Cross-Reactivity and/orImmunotherapeutic Effect of Proteins.

The methods described thus far have led to identification of epitopeareas on an acceptor protein, each containing epitope sequences. Thesesubsets of amino acids, are preferred for introducing mutations that aremeant to modify the immunogenecity of the acceptor protein. An even morepreferred subset of amino acids to target by mutagenesis are ‘hot spotamino acids’, which appear in several different epitope sequences, orwhich corresponds to anchor amino acids of the epitope patterns.

Thus, genetic engineering mutations should be designed in the epitopeareas, preferably in epitope sequences, and more preferably in the ‘hotspot amino acids’.

Substitution, Deletion, Insertion

When the epitope area(s) have been identified, a protein variantexhibiting a modified immunogenicity may be produced by changing theidentified epitope area of the parent protein by genetic engineeringmutation of a DNA sequence encoding the parent protein.

The epitope identified may be changed by substituting at least one aminoacid of the epitope area. In a preferred embodiment at least one anchoramino acid or hot spot amino acid is changed. The change will often besubstituting to an amino acid of different size, hydrophilicity, and/orpolarity, such as a small amino acid versus a large amino acid, ahydrophilic amino acid versus a hydrophobic amino acid, a polar aminoacid versus a non-polar amino acid and a basic versus an acidic aminoacid.

Other changes may be the addition/insertion or deletion of at least oneamino acid of the epitope sequence, preferably deleting an anchor aminoacid or a hot spot amino acid. Furthermore, an epitope pattern may bechanged by substituting some amino acids, and deleting/adding other.

In the claims a position to be changed by substitution, insertion,deletion will be indicated by: “Position xx to aaa, bbb, ccc, insertion,deletion”, meaning that position xx can be substituted by the amino acidaaa, bbb, ccc or that any amino acid can be inserted after position xxor that position xx can be deleted, e.g. “Position 27 to A, D, E,insertion, deletion” means that in position 27 the amino acid can besubstituted by A, D or E, or that any amino acid can be inserted afterposition 27, or that the amino acid in position 27 can be deleted.

When one uses protein engineering to eliminate epitopes, it is indeedpossible that new epitopes are created, or existing epitopes areduplicated. To reduce this risk, one can map the planned mutations at agiven position on the 3-dimensional structure of the protein ofinterest, and control the emerging amino acid constellation against adatabase of known epitope patterns, to rule out those possiblereplacement amino acids, which are predicted to result in creation orduplication of epitopes. Thus, risk mutations can be identified andeliminated by this procedure, thereby reducing the risk of makingmutations that lead to increased rather than decreased allergenicity.

Introduction of Residues for Chemical Derivatization in Epitope Areas

In yet another embodiment, one can design the mutation, such that aminoacids suitable for chemical modification are substituted for existingones in the epitope areas. The protein variant can then be conjugated toactivated polymers. Which amino acids to substitute and/or insert,depends in principle on the coupling chemistry to be applied. Thechemistry for preparation of covalent bioconjugates can be found in“Bioconjugate Techniques”, Hermanson, G. T. (1996), Academic Press Inc.,which is hereby incorporated as reference (see below). It is preferredto make conservative substitutions in the polypeptide when thepolypeptide has to be conjugated, as conservative substitutions securethat the impact of the substitution on the polypeptide structure islimited. In the case of providing additional amino groups this may bedone by substitution of arginine to lysine, both residues beingpositively charged, but only the lysine having a free amino groupsuitable as an attachment groups. In the case of providing additionalcarboxylic acid groups the conservative substitution may for instance bean asparagine to aspartic acid or glutamine to glutamic acidsubstitution. These residues resemble each other in size and shape,except from the carboxylic groups being present on the acidic residues.In the case of providing SH-groups the conservative substitution may bedone by changing threonine or serine to cysteine.

Chemical Conjugation

For chemical conjugation, the protein variant needs to be incubate withan active or activated polymer and subsequently separated from theunreacted polymer. This can be done in solution followed by purificationor it can conveniently be done using the immobilized protein variants,which can easily be exposed to different reaction environments andwashes.

In the case were polymeric molecules are to be conjugated with thepolypeptide in question and the polymeric molecules are not active theymust be activated by the use of a suitable technique. It is alsocontemplated according to the invention to couple the polymericmolecules to the polypeptide through a linker. Suitable linkers arewell-known to the skilled person. Methods and chemistry for activationof polymeric molecules as well as for conjugation of polypeptides areintensively described in the literature. Commonly used methods foractivation of insoluble polymers include activation of functional groupswith cyanogen bromide, periodate, glutaraldehyde, biepoxides,epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides,trichlorotriazine etc. (see R. F. Taylor, (1991), “Proteinimmobilisation. Fundamental and applications”, Marcel Dekker, N.Y.; S.S. Wong, (1992), “Chemistry of Protein Conjugation and Crosslinking”,CRC Press, Boca Raton; G. T. Hermanson et al., (1993), “ImmobilizedAffinity Ligand Techniques”, Academic Press, N.Y.). Some of the methodsconcern activation of insoluble polymers but are also applicable toactivation of soluble polymers e.g. periodate, trichlorotriazine,sulfonylhalides, divinylsulfone, carbodiimide etc. The functional groupsbeing amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on thepolymer and the chosen attachment group on the protein must beconsidered in choosing the activation and conjugation chemistry whichnormally consist of i) activation of polymer, ii) conjugation, and iii)blocking of residual active groups.

In the following a number of suitable polymer activation methods will bedescribed shortly. However, it is to be understood that also othermethods may be used.

Coupling polymeric molecules to the free acid groups of polypeptides maybe performed with the aid of diimide and for example amino-PEG orhydrazino-PEG (Pollak et al., (1976), J. Am. Chem. Soc., 98, 289-291) ordiazoacetate/amide (Wong et al., (1992), “Chemistry of ProteinConjugation and Crosslinking”, CRC Press).

Coupling polymeric molecules to hydroxy groups is generally verydifficult as it must be performed in water. Usually hydrolysispredominates over reaction with hydroxyl groups.

Coupling polymeric molecules to free sulfhydryl groups can be achievedwith special groups like maleimido or the ortho-pyridyl disulfide. Alsovinylsulfone (U.S. Pat. No. 5,414,135, (1995), Snow et al.) has apreference for sulfhydryl groups but is not as selective as the othermentioned.

Accessible arginine residues in the polypeptide chain may be targeted bygroups comprising two vicinal carbonyl groups.

Techniques involving coupling of electrophilically activated PEGs to theamino groups of Lysines may also be useful. Many of the usual leavinggroups for alcohols give rise to an amine linkage. For instance, alkylsulfonates, such as tresylates (Nilsson et al., (1984), Methods inEnzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p.56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135; Mosbach,K., Ed.; Academic Press: Orlando, pp. 65-79; Scouten et al., (1987),Methods in Enzymology vol. 135, Mosbach, K., Ed., Academic Press:Orlando, 1987; pp 79-84; Crossland et al., (1971), J. Amr. Chem. Soc.1971, 93, pp. 4217-4219), mesylates (Harris, (1985), supra; Harris etal., (1984), J. Polym. Sci. Polym. Chem. Ed. 22, pp 341-352), arylsulfonates like tosylates, and para-nitrobenzene sulfonates can be used.

Organic sulfonyl chlorides, e.g. Tresyl chloride, effectively convertshydroxy groups in a number of polymers, e.g. PEG, into good leavinggroups (sulfonates) that, when reacted with nucleophiles like aminogroups in polypeptides allow stable linkages to be formed betweenpolymer and polypeptide. In addition to high conjugation yields, thereaction conditions are in general mild (neutral or slightly alkalinepH, to avoid denaturation and little or no disruption of activity), andsatisfy the non-destructive requirements to the polypeptide.

Tosylate is more reactive than the mesylate but also less stabledecomposing into PEG, dioxane, and sulfonic acid (Zalipsky, (1995),Bioconjugate Chem., 6, 150-165). Epoxides may also been used forcreating amine bonds but are much less reactive than the abovementionedgroups.

Converting PEG into a chloroformate with phosgene gives rise tocarbamate linkages to Lysines. Essentially the same reaction can becarried out in many variants substituting the chlorine with N-hydroxysuccinimide (U.S. Pat. No. 5,122,614, (1992); Zalipsky et al., (1992),Biotechnol. Appl. Biochem., 15, p. 100-114; Monfardini et al., (1995),Bioconjugate Chem., 6, 62-69, with imidazole (Allen et al., (1991),Carbohydr. Res., 213, pp 309-319), with para-nitrophenol, DMAP (EP 632082 A1, (1993), Looze, Y.) etc. The derivatives are usually made byreacting the chloroformate with the desired leaving group. All thesegroups give rise to carbamate linkages to the peptide.

Furthermore, isocyanates and isothiocyanates may be employed, yieldingureas and thioureas, respectively.

Amides may be obtained from PEG acids using the same leaving groups asmentioned above and cyclic imid thrones (U.S. Pat. No. 5,349,001,(1994), Greenwald et al.). The reactivity of these compounds are veryhigh but may make the hydrolysis to fast.

PEG succinate made from reaction with succinic anhydride can also beused. The hereby comprised ester group make the conjugate much moresusceptible to hydrolysis (U.S. Pat. No. 5,122,614, (1992), Zalipsky).This group may be activated with N-hydroxy succinimide.

Furthermore, a special linker can be introduced. The most well studiedbeing cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252,3578-3581; U.S. Pat. No. 4,179,337, (1979), Davis et al.; Shafer et al.,(1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378.

Coupling of PEG to an aromatic amine followed by diazotation yields avery reactive diazonium salt, which can be reacted with a peptide insitu. An amide linkage may also be obtained by reacting an azlactonederivative of PEG (U.S. Pat. No. 5,321,095, (1994), Greenwald, R. B.)thus introducing an additional amide linkage.

As some peptides do not comprise many Lysines it may be advantageous toattach more than one PEG to the same Lysine. This can be done e.g. bythe use of 1,3-diamino-2-propanol.

PEGs may also be attached to the amino-groups of the enzyme withcarbamate linkages (WO 95/11924, Greenwald et al.). Lysine residues mayalso be used as the backbone.

The coupling technique used in the examples is the N-succinimidylcarbonate conjugation technique descried in WO 90/13590 (Enzon).

In a preferred embodiment, the activated polymer is methyl-PEG which hasbeen activated by N-succinimidyl carbonate as described WO 90/13590. Thecoupling can be carried out at alkaline conditions in high yields.

For coupling of polymers to the protein variants, it is preferred to useconditions similar to those described in WO 96/17929 and WO 99/00489(Novo Nordisk A/S) e.g. mono or bis activated PEG's of molecular weightranging from 100 to 5000 Da. For instance, a methyl-PEG 350 could beactivated with N-succinimidyl carbonate and incubated with proteinvariant at a molar ratio of more than 5 calculated as equivalents ofactivated PEG divided by moles of lysines in the protein of interest.For coupling to immobilized protein variant, the PEG:protein ratioshould be optimized such that the PEG concentration is low enough forthe buffer capacity to maintain alkaline pH throughout the reaction;while the PEG concentration is still high enough to ensure sufficientdegree of modification of the protein. Further, it is important that theactivated PEG is kept at conditions that prevent hydrolysis (i.e.dissolved in acid or solvents) and diluted directly into the alkalinereaction buffer. It is essential that primary amines are not presentother than those occurring in the lysine residues of the protein. Thiscan be secured by washing thoroughly in borate buffer. The reaction isstopped by separating the fluid phase containing unreacted PEG from thesolid phase containing protein and derivatized protein. Optionally, thesolid phase can then be washed with tris buffer, to block any unreactedsites on PEG chains that might still be present.

Introduction of Consensus Sequences for Post-Translational Modificationsin the Epitope Areas

In another embodiment, the mutations are designed, such that recognitionsites for post-translational modifications are introduced in the epitopeareas, and the protein variant is expressed in a suitable host organismcapable of the corresponding post-translational modification. Thesepost-translational modifications may serve to shield the epitope andhence lower the immunogenicity of the protein variant relative to theprotein backbone. Post-translational modifications includeglycosylation, phosphorylation, N-terminal processing, acylation,ribosylation and sulfatation. A good example is N-glycosylation.N-glycosylation is found at sites of the sequence Asn-Xaa-Ser,Asn-Xaa-Thr, or Asn-Xaa-Cys, in which neither the Xaa residue nor theamino acid following the tri-peptide consensus sequence is a proline (T.E. Creighton, ‘Proteins—Structures and Molecular Properties, 2ndedition, W.H. Freeman and Co., New York, 1993, pp. 91-93). It is thusdesirable to introduce such recognition sites in the sequence of thebackbone protein. The specific nature of the glycosyl chain of theglycosylated protein variant may be linear or branched depending on theprotein and the host cells. Another example is phosphorylation: Theprotein sequence can be modified so as to introduce serinephosphorylation sites with the recognition sequencearg-arg-(xaa)_(n)-ser (where n=0, 1, or 2) (SEQ ID NOS: 38 and 39),which can be phosphorylated by the cAMP-dependent kinase or tyrosinephosphorylation sites with the recognition sequence-lys/arg-(xaa)₃-asp/glu-(xaa)₃-tyr (SEC) ID NO: 40), which can usuallybe phosphorylated by tyrosine-specific kinases (T. E. Creighton,“Proteins—Structures and molecular properties”, 2nd ed., Freeman, N.Y.,1993).

Randomized Approaches to Introduce Modifications in Epitope Areas.

In order to generate protein variants, more than one amino acid residuemay be substituted, added or deleted, these amino acids preferably beinglocated in different epitope areas. In that case, it may be difficult toassess a priori how well the functionality of the protein is maintainedwhile antigenicity is reduced, especially since the possible number ofmutation-combinations becomes very large, even for a small number ofmutations. In that case, it will be an advantage, to establish a libraryof diversified mutants each having one or more changed amino acidsintroduced and selecting those variants, which show good retention offunction and at the same time a significant reduction in antigenicity.

A diversified library can be established by a range of techniques knownto the person skilled in the art (Reetz M T; Jaeger K E, in‘Biocatalysis—from Discovery to Application’ edited by Fessner W D, Vol.200, pp. 31-57 (1999); Stemmer, Nature, vol. 370, p. 389-391, 1994; Zhaoand Arnold, Proc. Natl. Acad. Sci., USA, vol. 94, pp. 7997-8000, 1997;or Yano et al., Proc. Natl. Acad. Sci., USA, vol. 95, pp 5511-5515,1998). These include, but are not limited to, ‘spiked mutagenesis’, inwhich certain positions of the protein sequence are randomized bycarring out PCR mutagenesis using one or more oligonucleotide primerswhich are synthesized using a mixture of nucleotides for certainpositions (Lanio T, Jeltsch A, Biotechniques, Vol. 25(6),958,962,964-965 (1998)). The mixtures of oligonucleotides used withineach triplet can be designed such that the corresponding amino acid ofthe mutated gene product is randomized within some predetermineddistribution function. Algorithms have been disclosed, which facilitatethis design (Jensen L J et al., Nucleic Acids Research, Vol. 26(3),697-702 (1998)).

In an embodiment substitutions are found by a method comprising thefollowing steps: 1) a range of substitutions, additions, and/ordeletions are listed encompassing several epitope areas (preferably inthe corresponding epitope sequences, anchor amino aids, and/or hotspots), 2) a library is designed which introduces a randomized subset ofthese changes in the amino acid sequence into the target gene, e.g. byspiked mutagenesis, 3) the library is expressed, and preferred variantsare selected. In another embodiment, this method is supplemented withadditional rounds of screening and/or family shuffling of hits from thefirst round of screening (J. E. Ness, et al, Nature Biotechnology, vol.17, pp. 893-896, 1999) and/or combination with other methods of reducingimmunogenicity by genetic means (such as that disclosed in WO 92/10755).

The library may be designed, such that at least one amino acid of theepitope area is substituted. In a preferred embodiment at least oneamino acid of the epitope sequence itself is changed, and in an evenmore preferred embodiment, one or more hot spot amino acids are changed.The library may be biased such that towards introducing an amino acid ofdifferent size, hydrophilicity, and/or polarity relative to the originalone of the ‘protein backbone’. For example changing a small amino acidto a large amino acid, a hydrophilic amino acid to a hydrophobic aminoacid, a polar amino acid to a non-polar amino acid or a basic to anacidic amino acid. Other changes may be the addition or deletion of atleast one amino acid of the epitope area, preferably deleting an anchoramino acid. Furthermore, substituting some amino acids and deleting oradding others may change an epitope.

Diversity in the protein variant library can be generated at the DNAtriplet level, such that individual codons are variegated e.g. by usingprimers of partially randomized sequence for a PCR reaction. Further,several techniques have been described, by which one can create alibrary with such diversity at several locations in the gene, which aretoo far apart to be covered by a single (spiked) oligonucleotide primer.These techniques include the use of in vivo recombination of theindividually diversified gene segments as described in WO 97/07205 onpage 3, line 8 to 29 or by using DNA shuffling techniques to create alibrary of full length genes that combine several gene segments each ofwhich are diversified e.g. by spiked mutagenesis (Stemmer, Nature 370,pp. 389-391, 1994 and U.S. Pat. Nos. 5,605,793 and 5,830,721). In thelatter case, one can use the gene encoding the “protein backbone” as atemplate double-stranded polynucleotide and combining this with one ormore single or double-stranded oligonucleotides as described in claim 1of U.S. Pat. No. 5,830,721. The single-stranded oligonucleotides couldbe partially randomized during synthesis. The double-strandedoligonucleotides could be PCR products incorporating diversity in aspecific region. In both cases, one can dilute the diversity withcorresponding segments containing the sequence of the backbone proteinin order to limit the number of changes that are on average introduced.As mentioned above, methods have been established for designing theratios of nucleotides (A; C; T; G) used at a particular codon duringprimer synthesis, so as to approximate a desired frequency distributionamong a set of desired amino acids at that particular codon. This allowsone to bias the partially randomized mutagenesis towards e.g.introduction of post-translational modification sites, chemicalmodification sites, or simply amino acids that are different from thosethat define the epitope or the epitope area. One could also approximatea sequence in a given location or epitope area to the correspondinglocation on a homologous, human protein.

Occasionally, one would be interested in testing a library that combinesa number of known mutations in different locations in the primarysequence of the ‘protein backbone’. These could be introducedpost-translational or chemical modification sites, or they could bemutations, which by themselves had proven beneficial for one reason oranother (e.g. decreasing antigenicity, or improving specific activity,performance, stability, or other characteristics). In such cases, it maybe desirable to create a library of diverse combinations of knownsequences. For example if 12 individual mutations are known, one couldcombine (at least) 12 segments of the ‘protein backbone’ gene in whicheach segment is present in two forms: one with and one without thedesired mutation. By varying the relative amounts of those segments, onecould design a library (of size 2¹²) for which the average number ofmutations per gene can be predicted. This can be a useful way ofcombining elements that by themselves give some, but not sufficienteffect, without resorting to very large libraries, as is often the casewhen using ‘spiked mutagenesis’. Another way to combine these ‘knownmutations’ could be by using family shuffling of oligomeric DNA encodingthe known changes with fragments of the full length wild type sequence.

Assays for Reduced Allergenicity

When protein variants have been constructed based on the methodsdescribed in this invention, it is desirable to confirm their antibodybinding capacity, functionality, immunogenicity and/or allergenicityusing a purified preparation. For that use, the protein variant ofinterest can be expressed in larger scale, purified by conventionaltechniques, and the antibody binding and functionality should beexamined in detail using dose-response curves and e.g. direct orcompetitive ELISA (C-ELISA).

The potentially reduced allergenicity (which is likely, but notnecessarily true for a variant w. low antibody binding) should be testedin in vivo or in vitro model systems: e.g. an in vitro assays forimmunogenicity such as assays based on cytokine expression profiles orother proliferation or differentiation responses of epithelial and othercells incl. B-cells and T-cells. Further, animal models for testingallergenicity should be set up to test a limited number of proteinvariants that show desired characteristics in vitro. Useful animalmodels include the guinea pig intratracheal model (GPIT) (Ritz, et al.Fund. Appl. Toxicol., 21, pp. 31-37, 1993), mouse subcutaneous(mouse-SC) (WO 98/30682, Novo Nordisk), the rat intratracheal (rat-IT)(WO 96/17929, Novo Nordisk), and the mouse intranasal (MINT) (Robinsonet al., Fund. Appl. Toxicol. 34, pp. 15-24, 1996) models.

The immunogenicity of the protein variant is measured in animal tests,wherein the animals are immunised with the protein variant and theimmune response is measured. Specifically, it is of interest todetermine the allergenicity of the protein variants by repeatedlyexposing the animals to the protein variant by the intratracheal routeand following the specific IgG and IgE titers. Alternatively, the mouseintranasal (MINT) test can be used to assess the allergenicity ofprotein variants. By the present invention the allergenicity is reducedat least 3 times as compared to the allergenicity of the parent protein,preferably 10 times reduced, more preferably 50 times.

However, the present inventors have demonstrated that the performance inELISA correlates closely to the immunogenic responses measured in animaltests. To obtain a useful reduction of the allergenicity of a protein,the IgE binding capacity of the protein variant must be reduced to atleast below 75%, preferably below 50%, more preferably below 25% of theIgE binding capacity of the parent protein as measured by theperformance in IgE ELISA, given the value for the IgE binding capacityof the parent protein is set to 100%.

Thus a first assessment of the immunogenicity and/or allergenicity of aprotein can be made by measuring the antibody binding capacity orantigenicity of the protein variant using appropriate antibodies. Thisapproach has also been used in the literature (WO 99/47680).

Assays for Altered Immunotherapeutic Effect

The immunotherapeutic effect of allergen vaccines can be assessed anumber of different ways. One is to measure the specific IgE binding,the reduction of which indicates a better allergen vaccine potential (WO99/47680, ALK-ABELLO). Also, several cellular assays could be employedto show the modified immuneresponse indicative of good allergen vaccinepotential as shown in several publications, all of which are herebyincorporated by reference (van Neerven et al, “T lymphocyte responses toallergens: Epitope-specificity and clinical relevance”, Immunol Today,1996, vol. 17, pp. 526-532; Hoffmann et al., Allergy, 1999, vol. 54, pp.446-454, WO 99/07880).

Eventually, clinical trials with allergic patients could be employedusing cellular or clinical end-point measurements. (Ebner et al., Clin.Exp. All., 1997, vol. 27, pp. 107-1015; Int. Arch. Allergy Immunol.,1999, vol. 119, pp 1-5).

Determining Functionality

A wide variety of protein functionality assays are available in theliterature. Especially, those suitable for automated analysis are usefulfor this invention. Several have been published in the literature suchas protease assays (WO 99/34011, Genencor International; J. E. Ness, etal, Nature Biotechn., 17, pp. 893-896, 1999), oxidoreductase assays(Chemy et al., Nature Biotechn., 17, pp. 379-384, 1999, and assays forseveral other enzymes (WO 99/45143, Novo Nordisk). Those assays thatemploy soluble substrates can be employed for direct analysis offunctionality of immobilized protein variants.

Cross-Reactivity

A related objective is to reduce cross-reactivity between ‘commercialallergens’ and ‘environmental allergens’. Cross-reactivities betweenfood allergens of different origin are well-known (Akkerdaas et al,Allergy 50, pp 215-220, 1995). Similarly, cross-reactivities betweenother environmental allergens (like pollen, dust mites etc.) andcommercial allergens (like enzyme proteins) have been established in theliterature (J. All. Clin. Immunol., 1998, vol. 102, pp. 679-686 and bythe present inventors. The molecular reason for this cross-reactivitycan be explored using epitope mapping. By finding epitope patterns usingantibodies raised against environmental allergen (donor protein) andmapping this information on a commercial allergen (the acceptorprotein), one may find the epitopes that are common to both proteins,and hence responsible for the cross-reactivity. Obviously, one can alsouse the commercial allergen as donor and the environmental allergen asacceptor. By modifying the commercial allergen using protein engineeringin the epitope areas identified as described above, one can reduce thecross-reactivity of the commercial allergen variant towards theenvironmental allergens (and vice versa). Hence, the use of the modifiedcommercial allergens would be safer than using the unmodified commercialallergen.

Testing of this approach would be done using an antibody-binding assaywith the protein variant (and its parent protein as control) andantibodies raised against the protein that cross-reacts with the parentprotein. The method is otherwise identical to those described in theMethods section for characterization of allergencitiy and antigenicity.

Wash Performance etc.

The modifications of the enzymes in the epitope areas as disclosed thepresent application may cause other effects to the enzyme than modifiedimmunogenicity. A modification may also change the performance of theenzyme, such as the wash performance, thermo stability, storagestability and increased catalytical activity of the enzyme.

The ability of an enzyme to catalyze the degradation of variousnaturally occurring substrates present on the objects to be cleanedduring e.g. wash is often referred to as its washing ability,wash-ability, detergency, or wash performance. Throughout thisapplication the term wash performance will be used to encompass thisproperty.

Commercial Enzyme Applications Industrial Applications

Another aspect of the invention is a composition comprising at least oneprotein (polypeptide) or enzyme of the invention. The composition maycomprise other polypeptides, proteins or enzymes and/or ingredientsnormally used in personal care products, such as shampoo, soap bars,skin lotion, skin creme, hair dye, toothpaste, household articles, agrochemicals, personal care products, such as cleaning preparations e.g.for contact lenses, cosmetics, toiletries, oral and dermalpharmaceuticals, compositions used for treating textiles, compositionsused for manufacturing food, e.g. baking, and feed etc.

Examples of said proteins(polypeptides)/enzymes include enzymesexhibiting protease, lipolytic enzyme, oxidoreductase, carbohydrase,transferase, such as transglutaminase, phytase and/or anti-microbialpolypeptide activity. These enzymes may be present as conjugates withreduced activity.

The protein of the invention may furthermore typically be used indetergent composition. It may be included in the detergent compositionin the form of a non-dusting granulate, a stabilized liquid, or aprotected enzyme. Non-dusting granulates may be produced, e.g., asdisclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 (both to NovoIndustri NS) and may optionally be coated by methods known in the art.Examples of waxy coating materials are poly(ethylene oxide) products(polyethylene glycol, PEG) with mean molecular weights of 1000 to 20000;ethoxylated nonylphenols having from 16 to 50 ethylene oxide units;ethoxylated fatty alcohols in which the alcohol contains from 12 to 20carbon atoms and in which there are 15 to 80 ethylene oxide units; fattyalcohols; fatty acids; and mono- and di- and triglycerides of fattyacids. Examples of film-forming coating materials suitable forapplication by fluid bed techniques are given in patent GB 1483591.Liquid enzyme preparations may, for instance, be stabilized by adding apolyol such as propylene glycol, a sugar or sugar alcohol, lactic acidor boric acid according to established methods. Other enzyme stabilizersare well known in the art. Protected enzymes may be prepared accordingto the method disclosed in EP 238,216.

The detergent composition may be in any convenient form, e.g. as powder,granules, paste or liquid. A liquid detergent may be aqueous, typicallycontaining up to 70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, each ofwhich may be anionic, nonionic, cationic, or zwitterionic. The detergentwill usually contain 0-50% of anionic surfactant such as linearalkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate(fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES),secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters,alkyl- or alkenylsuccinic acid, or soap. It may also contain 0-40% ofnonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylatedalcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. asdescribed in WO 92/06154).

The detergent composition may additionally comprise one or more otherenzymes, such as e.g. proteases, amylases, lipolytic enzymes, cutinases,cellulases, peroxidases, oxidases, and further anti-microbialpolypeptides.

The detergent may contain 1-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate, citrate,nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).The detergent may also be unbuilt, i.e. essentially free of detergentbuilder.

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylatessuch as polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine(TAED) or nonanoyloxybenzenesulfon-ate (NOBS). Alternatively, thebleaching system may comprise peroxyacids of, e.g., the amide, imide, orsulfone type.

The detergent composition of the invention comprising the polypeptide ofthe invention may be stabilized using conventional stabilizing agents,e.g. a polyol such as propylene glycol or glycerol, a sugar or sugaralcohol, lactic acid, boric acid, or a boric acid derivative such as,e.g., an aromatic borate ester, and the composition may be formulated asdescribed in, e.g., WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents,anti-soil-redeposition agents, dyes, bactericides, optical brighteners,or perfume.

The pH (measured in aqueous solution at use concentration) will usuallybe neutral or alkaline, e.g. in the range of 7-11.

Dishwashing Composition

Further, a modified enzyme according to the invention may also be usedin dishwashing detergents.

Dishwashing detergent compositions comprise a surfactant which may beanionic, non-ionic, cationic, amphoteric or a mixture of these types.The detergent will contain 0-90% of non-ionic surfactant such as low- tonon-foaming ethoxylated propoxylated straight-chain alcohols.

The detergent composition may contain detergent builder salts ofinorganic and/or organic types. The detergent builders may be subdividedinto phosphorus-containing and non-phosphorus-containing types. Thedetergent composition usually contains 1-90% of detergent builders.

Examples of phosphorus-containing inorganic alkaline detergent builders,when present, include the water-soluble salts especially alkali metalpyrophosphates, orthophosphates, and polyphosphates. An example ofphosphorus-containing organic alkaline detergent builder, when present,includes the water-soluble salts of phosphonates. Examples ofnon-phosphorus-containing inorganic builders, when present, includewater-soluble alkali metal carbonates, borates and silicates as well asthe various types of water-insoluble crystalline or amorphous aluminosilicates of which zeolites are the best-known representatives.

Examples of suitable organic builders include the alkali metal, ammoniumand substituted ammonium, citrates, succinates, malonates, fatty acidsulphonates, carboxymetoxy succinates, ammonium polyacetates,carboxylates, polycarboxylates, aminopolycarboxylates, polyacetylcarboxylates and polyhydroxsulphonates.

Other suitable organic builders include the higher molecular weightpolymers and co-polymers known to have builder properties, for exampleappropriate polyacrylic acid, polymaleic and polyacrylic/polymaleic acidcopolymers and their salts.

The dishwashing detergent composition may contain bleaching agents ofthe chlorine/bromine-type or the oxygen-type. Examples of inorganicchlorine/bromine-type bleaches are lithium, sodium or calciumhypochlorite and hypobromite as well as chlorinated trisodium phosphate.Examples of organic chlorine/bromine-type bleaches are heterocyclicN-bromo and N-chloro imides such as trichloroisocyanuric,tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids,and salts thereof with water-solubilizing cations such as potassium andsodium. Hydantoin compounds are also suitable.

The oxygen bleaches are preferred, for example in the form of aninorganic persalt, preferably with a bleach precursor or as a peroxyacid compound. Typical examples of suitable peroxy bleach compounds arealkali metal perborates, both tetrahydrates and monohydrates, alkalimetal percarbonates, persilicates and perphosphates. Preferred activatormaterials are TAED and glycerol triacetate.

The dishwashing detergent composition of the invention may be stabilizedusing conventional stabilizing agents for the enzyme(s), e.g. a polyolsuch as e.g. propylene glycol, a sugar or a sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g. an aromatic borate ester.

The dishwashing detergent composition of the invention may also containother conventional detergent ingredients, e.g. deflocculant material,filler material, foam depressors, anti-corrosion agents, soil-suspendingagents, sequestering agents, anti-soil redeposition agents, dehydratingagents, dyes, bactericides, fluorescers, thickeners and perfumes.

Finally, the enzyme of the invention may be used in conventionaldishwashing-detergents, e.g. in any of the detergents described in anyof the following patent publications: EP 518719, EP 518720, EP 518721,EP 516553, EP 516554, EP 516555, GB 2200132, DE 3741617, DE 3727911, DE4212166, DE 4137470, DE 3833047, WO 93/17089, DE 4205071, WO 52/09680,WO 93/18129, WO 93/04153, WO 92/06157, WO 92/08777, EP 429124, WO93/21299, U.S. Pat. No. 5,141,664, EP 561452, EP 561446, GB 2234980, WO93/03129, EP 481547, EP 530870, EP 533239, EP 554943, EP 346137, U.S.Pat. No. 5,112,518, EP 318204, EP 318279, EP 271155, EP 271156, EP346136, GB 2228945, CA 2006687, WO 93/25651, EP 530635, EP 414197, andU.S. Pat. No. 5,240,632.

Personal Care Applications

A particularly useful application area for low allergenic proteins or ofproteins with low cross-reactivity to environmental allergens would bein personal care products where the end-user is in close contact withthe protein, and where certain problems with allergenicity has beenencountered in experimental set-ups (Kelling et al., J. All. Clin. 1mm., 1998, Vol. 101, pp. 179-187 and Johnston et al., Hum. Exp.Toxicol., 1999, Vol. 18, p. 527).

First of all the conjugate or compositions of the invention canadvantageously be used for personal care products, such as hair care andhair treatment products. This include products such as shampoo, balsam,hair conditioners, hair waving compositions, hair dyeing compositions,hair tonic, hair liquid, hair cream, shampoo, hair rinse, hair spray.

Further contemplated are oral care products such as dentifrice, oralwashes, chewing gum.

Also contemplated are skin care products and cosmetics, such as skincream, skin milk, cleansing cream, cleansing lotion, cleansing milk,cold cream, cream soap, nourishing essence, skin lotion, milky lotion,calamine lotion, hand cream, powder soap, transparent soap, sun oil, sunscreen, shaving foam, shaving cream, baby oil lipstick, lip cream,creamy foundation, face powder, powder eye-shadow, powder, foundation,make-up base, essence powder, whitening powder.

Also for contact lenses hygiene products the conjugate of the inventioncan be used advantageously. Such products include cleaning anddisinfection products for contact lenses.

Proteases

Proteases are well-known active ingredients for cleaning of contactlenses. They hydrolyse the proteinaceous soil on the lens and therebymakes it soluble. Removal of the protein soil is essential for thewearing comfort.

Proteases are also effective ingredients in skin cleaning products,where they remove the upper layer of dead keratinaseous skin cells andthereby make the skin look brighter and fresher.

Proteases are also used in oral care products, especially for cleaningof dentures, but also in dentifrices.

Further, proteases are used in toiletries, bath and shower products,including shampoos, conditioners, lotions, creams, soap bars, toiletsoaps, and liquid soaps.

Lipolytic Enzymes

Lipolytic enzymes can be applied for cosmetic use as active ingredientsin skin cleaning products and anti-acne products for removal ofexcessive skin lipids, and in bath and shower products such as creamsand lotions as active ingredients for skin care.

Lipolytic enzymes can also be used in hair cleaning products (e.g.shampoos) for effective removal of sebum and other fatty material fromthe surface of hair.

Lipolytic enzymes are also effective ingredients in products forcleaning of contact lenses, where they remove lipid deposits from thelens surface.

Oxidoreductases

The most common oxidoreductase for personal care purposes is an oxidase(usually glucose oxidase) with substrate (e.g. glucose) that ensuresproduction of H₂O₂, which then will initiate the oxidation of forinstance SON⁻ or I⁻ into antimicrobial reagents (SCNO⁻ or I₂) by aperoxidase (usually lactoperoxidase). This enzymatic complex is known innature from e.g. milk and saliva.

It is being utilised commercially as anti-microbial system in oral careproducts (mouth rinse, dentifrice, chewing gum) where it also can becombined with an amyloglucosidase to produce the glucose. These systemsare also known in cosmetic products for preservation.

Anti-microbial systems comprising the combination of an oxidase and aperoxidase are know in the cleaning of contact lenses.

Another application of oxidoreductases is oxidative hair dyeing usingoxidases, peroxidases and laccases.

Free radicals formed on the surface of the skin (and hair) known to beassociated with the ageing process of the skin (spoilage of the hair).The free radicals activate chain reactions that lead to destruction offatty membranes, collagen, and cells. The application of free radicalscavengers such as Superoxide dismutase into cosmetics is well known (R.L. Goldemberg, DCI, Nov. 93, p. 48-52).

Protein disulfide isomerase (PDI) is also an oxidoreductase. It can beutilised for waving of hair (reduction and reoxidation of disulfidebonds in hair) and repair of spoiled hair (where the damage is mainlyreduction of existing disulfide bonds).

Carbohydrases

Plaque formed on the surface of teeth is composed mainly ofpolysaccharides. They stick to the surface of the teeth and themicroorganisms. The polysaccharides are mainly α-1,6 bound glucose(dextran) and α-1,3 bound glucose (mutan). The application of differenttypes of glucanases such as mutanase and dextranase helps hydrolysingthe sticky matrix of plaque, making it easier to remove by mechanicalaction.

Also other kinds of biofilm for instance the biofilm formed in lenscases can be removed by the action of glucanases.

Food and Feed

Further conjugated enzymes or polypeptides with reduced immunogenicityaccording to the invention may advantageously be used in themanufacturing of food and feed.

Proteases

The gluten in wheat flour is the essential ingredient responsible forthe ability of flour to be used in baked foodstuffs. Proteolytic enzymesare sometimes needed to modify the gluten phase of the dough, e.g. ahard wheat flour can be softened with a protease.

Neutrase® is a commercially available neutral metallo protease that canbe used to ensure a uniform dough quality and bread texture, and toimprove flavour. The gluten proteins are degraded either moderately ormore extensively to peptides, whereby close control is necessary inorder to avoid excessive softening of the dough.

Proteases are also used for modifying milk protein.

To coagulate casein in milk when producing cheese proteases such asrennet or chymosin may be used.

In the brewery industry proteases are used for brewing with unmaltedcereals and for controlling the nitrogen content.

In animal feed products proteases are used so to speak to expand theanimals digestion system.

Lipolytic Enzymes

Addition of lipolytic enzyme results in improved dough properties and animproved breadmaking quality in terms of larger volume, improved crumbstructure and whiter crumb colour. The observed effect can be explainedby a mechanism where the lipolytic enzyme changes the interactionbetween gluten and some lipids fragment during dough mixing. Thisresults in an improved gluten network.

The flavour development of blue roan cheese (e.g. Danablue), certainItalian type cheese, and other dairy products containing butter-fat, aredependent on the degradation of milk fat into free fatty acids.Lipolytic enzymes may be used for developing flavour in such products.

In the oil- and fat producing industry lipases are used e.g. to minimizethe amount of undesirable side-products, to modify fats byinteresterification, and to synthesis of esters.

Oxidoreductases

Further oxidoreductases with reduced immunogenicity according to theinvention may advantageously be used in the manufacturing of food andfeed.

Several oxidoreductases are used for baking, glucose oxidase,lipoxygenase, peroxidase, catalase and combinations hereof.Traditionally, bakers strengthen gluten by adding ascorbic acid andpotassium bromate. Some oxidoreductases can be used to replace bromatein dough systems by oxidation of free sulfydryl units in glutenproteins. Hereby disulphide linkages are formed resulting in stronger,more elastic doughs with greater resistance.

Gluzyme™ (Novozymes A/S) is a glucose oxidase preparation with catalaseactivity that can be used to replace bromate. The dough strengthen ismeasured as greater resistance to mechanical shock, better oven springand larger loaf volume.

Carbohydrases

Flour has varying content of amylases leading to differences in thebaking quality. Addition of amylases can be necessary in order tostandardize the flour. Amylases and pentosanases generally provide sugarfor the yeast fermentation, improve the bread volume, retardretrogradation, and decrease the staling rate and stickiness thatresults from pentosan gums. Examples of carbohydrases are given below.

Certain maltogenic amylases can be used for prolonging the shelf life ofbread for two or more days without causing gumminess in the product.Selectively modifies the gelatinized starch by cleaving from thenon-reducing end of the starch molecules, low molecular wight sugars anddextrins. The starch is modified in such a way that retrogradation isless likely to occur. The produced low-molecular-weight sugars improvethe baked goods water retention capacity without creating theintermediate-length dextrins that result in gumminess in the finishedproduct. The enzyme is inactivated during bread baking, so it can beconsidered a processing aid that does not have to be declared on thelabel. Overdosing of Novamyl can almost be excluded.

The bread volume can be improved by fungal α-amylases which furtherprovide good and uniform structure of the bread crumb. Said α-amylasesare endoenzymes that produce maltose, dextrins and glucose. Cereal andsome bacterial α-amylases are inactivated at temperatures above thegelatinization temperature of starch, therefore when added to wheatdough it results in a low bread volume and a sticky bread interior.Fungamyl has the advantage of being thermolabile and is inactivated justbelow the gelatinization temperature.

Enzyme preparations containing a number of pentosanase andhemi-cellulase activities can improve the handling and stability of thedough, and improves the freshness, the crumb structure and the volume ofthe bread.

By hydrolysing the pentosans fraction in flour, it will lose a greatdeal of its water-binding capacity, and the water will then be availablefor starch and gluten. The gluten becomes more pliable and extensible,and the starch gelatinizes more easily. Pentosanases can be used incombination with or as an alternative to emulsifiers.

Further carbohydrases are user for producing syrups from starch, whichare widely used in soft drinks, sweets, meat products, dairy products,bread products, ice cream, baby food, jam etc.

The conversion of starch is normally carried out three steps. First thestarch is liquefied, by the use of alpha-amylases. Maltodextrins,primary consisting of oligosaccharides and dextrins, are obtained.

The mixture is then treated with an amyloglucosidase for hydrolysing theoligosaccharides and dextrins into glucose. This way a sweeter productis obtained. If high maltose syrups are desired beta-amylases alone orin combination with a pullulanase (de-branching enzyme) may be used.

The glucose mixture can be made even sweeter by isomerization tofructose. For this an immobilized glucose isomerase can be used.

In the sugar industry, it is common practice to speed up the break downof present starch in cane juices. Thereby the starch content in the rawsugar is reduced and filtration at the refinery facilitated.

Furthermore dextranases are used to break down dextran in raw sugarjuices and syrups.

In the alcohol industry alpha-amylases is advantageously being used forthinning of starch in distilling mashes.

In the brewing industry alpha-amylases is used for adjunct liquefaction.

In the dairy industry beta-galactosidases (lactase) is used whenproducing low lactose milk for persons suffering from lactosemalabsorption.

When flavoured milk drinks are produced from lactase-treated milk, theaddition of sugar can be reduced without reducing the sweetness of theproduct.

In the production of condensed milk, lactose crystallization can beavoided by lactase treatment, and the risk of thickening caused bycasein coagulation in lactose crystals is thus reduced.

When producing ice cream made from lactase-treated milk (or whey) nolactose crystals will be formed and the defect, sandiness, will notoccur.

Further, xylanases are known to be used within a number of food/feedindustrial applications as described in WO 94/21785 (Novo Nordisk A/S).

Alpha-amylases are used in the animal feed industry to be added tocereal-containing feed to improve the digestibility of starch.

Anti-Microbial Polypeptides

Certain bacteriolytic enzymes may be used e.g. to wash carcasses in themeat packing industry (see U.S. Pat. No. 5,354,681 from Novo IndustriA/S)

Transferases

Transglutaminases with reduced immunogenicity according to the inventionmay advantageously be used in the manufacturing of food and feed.

Transglutaminases has the ability to crosslinking protein.

This property can be used for gelling of aqueous phases containingproteins. This may be used for when producing of spreads (DK patentapplication no. 1071/84 from Novo Nordisk A/S).

Transglutaminases are being used for improvement of baking quality offlour e.g. by modifying wheat flour to be used in the preparation ofcakes with improved properties, such as improved taste, dent, mouth-feeland a higher volume (see JP 1-110147).

Further producing paste type food material e.g. used as fat substitutionin foods as ice cream, toppings, frozen desserts, mayonnaises and lowfat spreads (see WO 93/22930 from Novo Nordisk A/S).

Furthermore for preparation of gels for yoghurt, mousses, cheese,puddings, orange juice, from milk and milk-like products, and binding ofchopped meat product, improvement of taste and texture of food proteins(see WO 94/21120 and WO 94/21129 from Novo Nordisk A/S).

Phytases

Phytases of the invention may advantageously be used in themanufacturing of food, such as breakfast cereal, cake, sweets, drinks,bread or soup etc., and animal feed.

Phytases may be used either for exploiting the phosphorus bound in thephytate/phytic acid present in vegetable protein sources or forexploiting the nutritionally important minerals bound in phytic acidcomplexes.

Microbial phytase may be added to feedstuff of monogastric animals inorder to avoid supplementing the feed with inorganic phosphorus (seeU.S. Pat. No. 3,297,548).

Further phytases may be used in soy processing. Soyabean meal maycontain high levels of the anti-nutritional factor phytate which rendersthis protein source unsuitable for application in baby food and feed forfish, calves and other non-ruminants, since the phytate chelatesessential minerals present therein (see EP 0 420 358).

Also for baking purposes phytases may be used. Bread with better qualitycan be prepared by baking divided pieces of a dough containing wheatflour etc. and phytase (see JP-0-3076529-A).

A high phytase activity as in koji mold are known to be used forproducing refined sake (see JP-0-6070749-A).

Textile Applications Proteases

Proteases are used for degumming and sand washing of silk.

Lipolytic Enzymes

Lipolytic enzymes are used for removing fatty matter containinghydrophobic esters (e.g. triglycerides) during the finishing of textiles(see e.g. WO 93/13256 from Novo Nordisk A/S).

Oxidoreductases

In bleach clean up of textiles catalases may serve to remove excesshydrogen peroxide.

Carbohydrases

Cellulolytic enzymes are widely used in the finishing of denim garmentsin order to provide a localized variation in the colour density of thefabric (Enzyme facilitated “stone wash”).

Also cellulolytic enzymes find use in the bio-polishing process.Bio-Polishing is a specific treatment of the yarn surface which improvesfabric quality with respect to handle and appearance without loss offabric wettability. Bio-polishing may be obtained by applying the methoddescribed e.g. in WO 93/20278.

During the weaving of textiles, the threads are exposed to considerablemechanical strain. In order to prevent breaking, the threads are usuallyreinforced by the coating (sizing) with a gelatinous substance (size).The most common sizing agent is starch in native or modified form. Auniform and durable finish can thus be obtained only after removal ofthe size from the fabric, the so-called desizing. Desizing of fabricssized with a size containing starch or modified starch is preferablyfacilitated by use of amylolytic enzymes.

Oral and Dermal Pharmaceuticals Proteases

Different combinations of highly purified proteases (e.g. Trypsin andChymotrypsin) are used in pharmaceuticals to be taken orally, and dermalpharmaceuticals for combating e.g. inflammations, edemata and injuries.

Leather Production Transferase

Transglutaminase is known to be used to casein-finishing leather byacting as a hardening agent (see WO 94/13839 from Novo Nordisk).

Hard Surface Cleaning

Cleaning of hard surfaces e.g. in the food industry is often difficult,as equipment used for producing dairies, meat, sea food products,beverages etc. often have a complicated shape. The use of surfactantcompositions in the form gels and foams comprising enzymes have shown tofacilitate and improve hard surface cleaning. Enzymes, whichadvantageously may be added in such surfactant compositions, are inparticular proteases, lipolytic enzymes, amylases and cellulases.

Such hard surface cleaning compositions comprising enzymes may alsoadvantageously be used in the transport sector, for instance for washingcars and for general vessel wash.

Furthermore this invention relates to the method by which the proteinvariants are being synthesised and expressed in host cells. This isachieved by culturing host cells capable of expressing a polypeptide ina suitable culture medium to obtain expression and secretion of thepolypeptide into the medium, followed by isolation of the polypeptidefrom the culture medium. The host cell may be any cell suitable for thelarge-scale production of proteins, capable of expressing a protein andbeing transformed by an expression vector.

The host cell comprises a DNA construct as defined above, optionally thecells may be transformed with an expression vector comprising a DNAconstruct as defined above. The host cell is selected from any suitablecell, such as a bacterial cell, a fungal cell, an animal cell, such asan insect cell or a mammalian cell, or a plant cell.

Immunotherapy

A number of vaccination approaches have been described to for infectivediseases as well as for non-infective diseases (such as cancers). In anumber of cases, the antigen provided is an isolated protein orprotein-adjuvant mixture and more and more often, the protein isrecombinant (e.g. the hepatitits B vaccine from Merck & Co). In thesecases, it could be desirable to modify the immunogenicity of the antigenvaccine, such that it offers a stronger or more specific protection.This can be achieved by protein engineering of the amino acid sequenceof the antigen, and would be greatly facilitated by the use of themethods of this invention for identification of epitopes on the antigenvaccine to be the favored sites for modification.

There are several examples of vaccine molecules that have beenengineered to achieve a specific immune protection against virus,parasites or cancer (Ryu and Nam, Biotechnol. Prog., 2000, vol. 16 pp.2-16; and references cited therein). “The goal is often to vaccinatewith a minimal strucutre consisting of a well-defined antigen, tostimulate an effective specific immune response, while avoidingpotentially hazardous risks” (Ryu and Nam, Biotechnol. Prog., 2000, vol.16 pp. 2-16). Thus, the methods of this invention can be used toidentify such minimal structures that define an antigen (or epitopethereof) whether in the form of the parent protein scaffold with anumber of mutations introduced in it, or whether it is in the form ofthe antibody binding peptides themselves.

Allergen Vaccines

Today, a patient suffering allergic disease may be subjected to allergyvaccine therapy using allergens selected on the basis of testing thespecificity of the patient's serum IgE against a bank of allergenextracts (or similar specificity tests of the patient's sensibilizationsuch as skin prick test.

One could improve the quality of characterization by using antibodybinding peptides corresponding to various epitope sequences on theprotein allergens of interest. This would require a kit comprisingreagents for such specificity characterization, e.g. the antibodybinding peptides of desired specificity. It would be preferred to useantibody binding sequences in the kit, which correspond to definedepitope sequences known to be specific for the allergen underinvestigation (i.e. not identified on other allergens and/or notcross-reacting with sera raised against other allergens). This kit wouldbe useful to specifying which allergy the patient is suffering from.This kit will lead to a more specific answer than those kits used today,and hence to a better selection of allergen vaccine therapy for theindividual patient.

Further, the knowledge about cross-reacting epitopes may improve vaccinedevelopment.

In an extension of this approach, one could also characterize thepatient's serum by identifying the corresponding antibody bindingpeptides among a random display library using the aforementionedmethods. This again may lead to a better selection of allergen vaccinetherapy.

Further, one could use the individual antibody binding sequences asallergen vaccines leading to more specific allergen vaccine. Theseantibody binding sequences could be administered in an isolated form orfused to a membrane protein of the phage display system, or to anotherprotein, which may have beneficial effect for the immunoprotectiveeffect of the antibody binding peptide (Dalum et al., NatureBiotechnology, 1999, Vol. 17, pp. 666-669).

D) Variations Possible Parent Protein

The “parent protein” can in principle be any protein molecule ofbiological origin, non-limiting examples of which are peptides,polypeptides, proteins, enzymes, post-translationally modifiedpolypeptides such as lipopeptides or glycosylated peptides,anti-microbial peptides or molecules, and proteins having pharmaceuticalproperties etc.

Accordingly the invention relates to a method, wherein the “parentprotein” is chosen from the group consisting of polypeptides, smallpeptides, lipopeptides, antimicrobials, and pharmaceutical polypeptides.

The term “pharmaceutical polypeptides” is defined as polypeptides,including peptides, such as peptide hormones, proteins and/or enzymes,being physiologically active when introduced into the circulatory systemof the body of humans and/or animals.

Pharmaceutical polypeptides are potentially immunogenic as they areintroduced into the circulatory system.

Examples of “pharmaceutical polypeptides” contemplated according to theinvention include insulin, ACTH, glucagon, somatostatin, somatotropin,thymosin, parathyroid hormone, pigmentary hormones, somatomedin,erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalmicreleasing factors, antidiuretic hormones, thyroid stimulating hormone,relaxin, interferon, thrombopoietin (TPO) and prolactin.

However, the proteins are preferably to be used in industry,housekeeping and/or medicine, such as proteins used in personal careproducts (for example shampoo; soap; skin, hand and face lotions; skin,hand and face cremes; hair dyes; toothpaste), food (for example in thebaking industry), detergents and pharmaceuticals.

Antimicrobial Peptides.

The antimicrobial peptide (AMP) may be, e.g., a membrane-activeantimicrobial peptide, or an antimicrobial peptide affecting/interactingwith intracellular targets, e.g. binding to cell DNA. The AMP isgenerally a relatively short peptide, consisting of less than 100 aminoacid residues, typically 20-80 residues. The antimicrobial peptide hasbactericidal and/or fungicidal effect, and it may also have antiviral orantitumour effects. It generally has low cytotoxicity against normalmammalian cells.

The antimicrobial peptide is generally highly cationic and hydrophobic.It typically contains several arginine and lysine residues, and it maynot contain a single glutamate or asparatate. It usually contains alarge proportion of hydrophobic residues. The peptide generally has anamphiphilic structure, with one surface being highly positive and theother hydrophobic.

The bioactive peptide and the encoding nucleotide sequence may bederived from plants, invertebrates, insects, amphibians and mammals, orfrom microorganisms such as bacteria and fungi.

The antimicrobial peptide may act on cell membranes of targetmicroorganisms, e.g. through nonspecific binding to the membrane,usually in a membrane-parallel orientation, interacting only with oneface of the bilayer.

The antimicrobial peptide typically has a structure belonging to one offive major classes: a helical, cystine-rich (defensin-like), b-sheet,peptides with an unusual composition of regular amino acids, andpeptides containing uncommon modified amino acids.

Examples of alpha-helical peptides are Magainin 1 and 2; Cecropin A, Band P1; CAP18; Andropin; Clavanin A or AK; Styelin D and C; and BuforinII. Examples of cystine-rich peptides are a-Defensin HNP-1 (humanneutrophil peptide) HNP-2 and HNP-3; b-Defensin-12, Drosomycin,g1-purothionin, and Insect defensin A. Examples of b-sheet peptides areLactoferricin B, Tachyplesin I, and Protegrin PG1-5. Examples ofpeptides with an unusual composition are Indolicidin; PR-39; BactenicinBac5 and Bac7; and Histatin 5. Examples of peptides with unusual aminoacids are Nisin, Gramicidin A, and Alamethicin.

Another example is the antifungal peptide (AFP) from Aspergillusgiganteus. As explained in detail in WO 94/01459, which is herebyincorporated by reference, the antifungal polypeptide having the aminoacid sequence shown in FIG. 1 has been found in several strains of thefungal species A. giganteus, an example of which is the A. giganteusstrain deposited with the Centraallbureau voor Schimmelcultures (CBS)under the deposition number CBS 526.65.

However, the antifungal polypeptide, or variants thereof, suitable forthe use according to the invention are expected to be derivable fromother fungal species, especially other Aspergillus species such as A.pallidus, A. clavatus, A. longivesica, A. rhizopodus and A.clavatonanicus, because of the close relationship which exists betweenthese species and A. giganteus.

In one embodiment of the invention the protein is an enzyme, such asglycosyl hydrolases, carbohydrases, peroxidases, proteases, lipolyticenzymes, phytases, polysaccharide lyases, oxidoreductases,transglutaminases and glycoseisomerases, in particular the following.

Parent Proteases

Parent proteases (i.e. enzymes classified under the EnzymeClassification number E.C. 3.4 in accordance with the Recommendations(1992) of the International Union of Biochemistry and Molecular Biology(IUBMB)) include proteases within this group.

Examples include proteases selected from those classified under theEnzyme Classification (E.C.) numbers:

3.4.11 (i.e. so-called aminopeptidases), including 3.4.11.5 (Prolylaminopeptidase), 3.4.11.9 (X-pro aminopeptidase), 3.4.11.10 (Bacterialleucyl aminopeptidase), 3.4.11.12 (Thermophilic aminopeptidase),3.4.11.15 (Lysyl aminopeptidase), 3.4.11.17 (Tryptophanylaminopeptidase), 3.4.11.18 (Methionyl aminopeptidase).

3.4.21 (i.e. so-called serine endopeptidases), including 3.4.21.1(Chymotrypsin), 3.4.21.4 (Trypsin), 3.4.21.25 (Cucumisin), 3.4.21.32(Brachyurin), 3.4.21.48 (Cerevisin) and 3.4.21.62 (Subtilisin);

3.4.22 (i.e. so-called cysteine endopeptidases), including 3.4.22.2(Papain), 3.4.22.3 (Ficain), 3.4.22.6 (Chymopapain), 3.4.22.7(Asclepain), 3.4.22.14 (Actimidain), 3.4.22.30 (Caricain) and 3.4.22.31(Ananain);

3.4.23 (i.e. so-called aspartic endopeptidases), including 3.4.23.1(Pepsin A), 3.4.23.18 (Aspergillopepsin I), 3.4.23.20 (Penicillopepsin)and 3.4.23.25 (Saccharopepsin); and

3.4.24 (i.e. so-called metalloendopeptidases), including 3.4.24.28(Bacillolysin).

Serine Proteases

A serine protease is an enzyme which catalyzes the hydrolysis of peptidebonds, and in which there is an essential serine residue at the activesite (White, Handler and Smith, 1973 “Principles of Biochemistry,” FifthEdition, McGraw-Hill Book Company, NY, pp. 271-272).

The bacterial serine proteases have molecular weights in the 20,000 to45,000 Dalton range. They are inhibited by diisopropylfluorophosphate.They hydrolyze simple terminal esters and are similar in activity toeukaryotic chymotrypsin, also a serine protease. A more narrow term,alkaline protease, covering a sub-group, reflects the high pH optimum ofsome of the serine proteases, from pH 9.0 to 11.0 (for review, seePriest (1977) Bacteriological Rev. 41 711-753).

Subtilases

A sub-group of the serine proteases tentatively designated subtilaseshas been proposed by Siezen et al., Protein Engng. 4 (1991) 719-737 andSiezen et al. Protein Science 6 (1997) 501-523. They are defined byhomology analysis of more than 170 amino acid sequences of serineproteases previously referred to as subtilisin-like proteases. Asubtilisin was previously often defined as a serine protease produced byGram-positive bacteria or fungi, and according to Siezen et al. now is asubgroup of the subtilases. A wide variety of subtilases have beenidentified, and the amino acid sequence of a number of subtilases hasbeen determined. For a more detailed description of such subtilases andtheir amino acid sequences reference is made to Siezen et al., (1997).

Savinase-Like Subtilisin

One subgroup of the subtilases may be classified as savinase-likesubtilisins, having at least 81% homology to Savinase, preferably atleast 85% homology, more preferably at least 90% homology, even morepreferably at least 96% homology, most preferably at least 98% homologyto Savinase.

Parent Subtilase

The term “parent subtilase” describes a subtilase defined according toSiezen et al. (1991 and 1997). For further details see description of“SUBTILASES” immediately above. A parent subtilase may also be asubtilase isolated from a natural source, wherein subsequentmodifications have been made while retaining the characteristic of asubtilase. Furthermore, a parent subtilase may also be a subtilase whichhas been prepared by the DNA shuffling technique, such as described byJ. E. Ness et al., Nature Biotechnology, 17, 893-896 (1999).

Alternatively the term “parent subtilase” may be termed “wild typesubtilase”.

Modification(s) of a Subtilase Variant

The term “modification(s)” used herein is defined to include chemicalmodification of a subtilase as well as genetic manipulation of the DNAencoding a subtilase. The modification(s) can be replacement(s) of theamino acid side chain(s), substitution(s), deletion(s) and/or insertionsin or at the amino acid(s) of interest.

Subtilase Variant

In the context of this invention, the term subtilase variant or mutatedsubtilase means a subtilase that has been produced by an organism whichis expressing a mutant gene derived from a parent microorganism whichpossessed an original or parent gene and which produced a correspondingparent enzyme, the parent gene having been mutated in order to producethe mutant gene from which said mutated subtilase protease is producedwhen expressed in a suitable host.

Examples of relevant subtilisins comprise subtilisin BPN′, subtilisinamylosacchariticus, subtilisin 168, subtilisin mesentericopeptidase,subtilisin Carlsberg, subtilisin DY, subtilisin 309, subtilisin 147,PD498 (WO 93/24623), thermitase, aqualysin, Bacillus PB92 protease,proteinase K, Protease TW7, and Protease TW3.

Preferred commercially available protease enzymes include Alcalase™,Savinase™ Primase™, Duralase™, Neutrase®, Dyrazym®, Esperase™, Pyrase®,Pancreatic Trypsin NOVO (PTN), Bio-Feed™ Pro, Clear-Lens Pro, andRelase® (Novozymes A/S), Maxatase™ Maxacal™, Maxapem™, Properase™,Purafect™, Purafect OxP™ (Genencor International Inc.).

It is to be understood that also protease variants are contemplated asthe parent protease. Examples of such protease variants are disclosed inEP 130.756 (Genentech), EP 214.435 (Henkel), WO 87/04461 (Amgen), WO87/05050 (Genex), EP 251.446 (Genencor), EP 260.105 (Genencor), Thomaset al., (1985), Nature. 318, p. 375-376, Thomas et al., (1987), J. Mol.Biol., 193, pp. 803-813, Russel et al., (1987), Nature, 328, p. 496-500,WO 88/08028 (Genex), WO 88/08033 (Amgen), WO 89/06279 (Novo NordiskA/S), WO 91/00345 (Novo Nordisk A/S), EP 525 610 (Solvay) and WO94/02618 (Gist-Brocades N.V.).

The activity of proteases can be determined as described in “Methods ofEnzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim, vol.5.

Parent Lipolytic Enzymes

Lipolytic enzymes are classified in EC 3.1.1 Carboxylic Ester Hydrolasesaccording to Enzyme Nomenclature (available athttp://www.chem.qmw.ac.uk/iubmb/enzyme). The lipolytic enzyme may have asubstrate specificity with an activity such as EC 3.1.1.3triacylglycerol lipase, EC 3.1.1.4 phospholipase A2, EC 3.1.1.5lysophospholipase, EC 3.1.1.26 galactolipase, EC 3.1.1.32 phospholipaseA1, EC 3.1.1.73 feruloyl esterase or EC 3.1.1.74 cutinase.

The parent lipolytic enzyme may be prokaryotic, particularly a bacterialenzyme, e.g. from Pseudomonas. Examples are Pseudomonas lipases, e.g.from P. cepacia (U.S. Pat. No. 5,290,694, pdb file 1OIL), P. glumae (NFrenken et al. (1992), Appl. Envir. Microbiol. 58 3787-3791, pdb files1TAH and 1QGE), P. pseudoalcaligenes (EP 334 462) and Pseudomonas sp.strain SD 705 (FERM BP-4772) (WO 95/06720, EP 721 981, WO 96/27002, EP812 910). The P. glumae lipase sequence is identical to the amino acidsequence of Chromobacterium viscosum (DE 3908131 A1). Other examples arebacterial cutinases, e.g. from Pseudomonas such as P. mendocina (U.S.Pat. No. 5,389,536) or P. putida (WO 88/09367).

Alternatively, the parent lipolytic enzyme may be eukaryotic, e.g. afungal lipolytic enzyme such as lipolytic enzymes of the Humicola familyand the Zygomycetes family and fungal cutinases.

Examples of fungal cutinases are the cutinases of Fusarium solani pisi(S. Longhi et al., Journal of Molecular Biology, 268 (4), 779-799(1997)) and Humicola insolens (U.S. Pat. No. 5,827,719).

The parent lipolytic enzyme may be fungal and may have an amino acidsequence that can be aligned with SEQ ID NO: 1 which is the amino acidsequence shown in positions 1-269 of SEQ ID NO: 2 of U.S. Pat. No.5,869,438 for the lipase from Thermomyces lanuginosus (synonym Humicolalanuginosa), described in EP 258 068 and EP 305 216 (trade nameLIPOLASE). The parent lipolytic enzyme may particularly have an aminoacid sequence with at least 50% homology with SEQ ID NO: 1. In additionto the lipase from T. lanuginosus, other examples are a lipase fromPenicillium camembertii (P25234), a lipase from Fusasrium,lipase/phospholipase from Fusarium oxysporum (EP 130064, WO 98/26057),lipase from F. heterosporum (R87979), lysophospholipase from Aspergillusfoetidus (W33009), phospholipase A1 from A. oryzae (JP-A 10-155493),lipase from A. oryzae (D85895), lipase/ferulic acid esterase from A.niger (Y09330), lipase/ferulic acid esterase from A. tubingensis(Y09331), lipase from A. tubingensis (WO 98/45453), lysophospholipasefrom A. niger (WO 98/31790), lipase from F. solanii having anisoelectric point of 6.9 and an apparent molecular weight of 30 kDa (WO96/18729).

Other examples are the Zygomycetes family of lipases comprising lipaseshaving at least 50% homology with the lipase of Rhizomucor miehei(P19515. This family also includes the lipases from Absidia reflexa, A.sporophora, A. corymbifera, A. blakesleeana, A. griseola (all describedin WO 96/13578 and WO 97/27276) and Rhizopus oryzae (P21811). Numbers inparentheses indicate publication or accession to the EMBL, GenBank,GeneSeqp or Swiss-Prot databases.

Examples of lipases include lipases derived from the followingmicroorganisms. The indicated patent publications are incorporatedherein by reference:

Humicola, e.g. H. brevispora, H. brevis var. thermoidea.

Pseudomonas, e.g. Ps. fragi, Ps. stutzeri, Ps. cepacia and Ps.fluorescens (WO 89/04361), or Ps. plantarii or Ps. gladioli (U.S. Pat.No. 4,950,417 (Solvay enzymes)) or Ps. alcaligenes and Ps.pseudoalcaligenes (EP 218 272) or.

Candida, e.g. C. cylindracea (also called C. rugosa) or C. antarctica(WO 88/02775) or C. antarctica lipase A or B (WO 94/01541 and WO89/02916).

Geotricum, e.g. G. candidum (Schimada et al., (1989), J. Biochem., 106,383-388).

Rhizopus, e.g. R. delemar (Hass et al., (1991), Gene 109, 107-113) or R.niveus (Kugimiya et al., (1992) Biosci. Biotech. Biochem 56, 716-719) orR. oryzae.

Bacillus, e.g. B. subtilis (Dartois et al., (1993) Biochemica etBiophysica acta 1131, 253-260) or B. stearothermophilus (JP 64/7744992)or B. pumilus (WO 91/16422).

Specific examples of readily available commercial lipases includeLipolase® (WO 98/35026) Lipolase™ Ultra, Lipozyme®, Palatase®, Novozym®435, Lecitase® (all available from Novozymes A/S).

Examples of other lipases are Lumafast™, Ps. mendocian lipase fromGenencor Int. Inc.; Lipomax™, Ps. pseudoalcaligenes lipase from GistBrocades/Genencor Int. Inc.; Fusarium solani lipase (cutinase) fromUnilever; Bacillus sp. lipase from Solvay enzymes. Other lipases areavailable from other companies.

It is to be understood that also lipase variants are contemplated as theparent enzyme. Examples of such are described in e.g. WO 93/01285 and WO95/22615.

The activity of the lipase can be determined as described in “Methods ofEnzymatic Analysis”, Third Edition, 1984, Verlag Chemie, Weinhein, vol.4, or as described in AF 95/5 GB (available on request from NovozymesA/S).

Parent Oxidoreductases

Parent oxidoreductases (i.e. enzymes classified under the EnzymeClassification number E.C. 1 (Oxidoreductases) in accordance with theRecommendations (1992) of the International Union of Biochemistry andMolecular Biology (IUBMB)) include oxidoreductases within this group.

Examples include oxidoreductases selected from those classified underthe Enzyme Classification (E.C.) numbers:

Glycerol-3-phosphate dehydrogenase (NAD) (1.1.1.8), Glycerol-3-phosphatedehydrogenase [NAD(P)] (1.1.1.94), Glycerol-3-phosphate 1-dehydrogenase[NADP] (1.1.1.94), Glucose oxidase (1.1.3.4), Hexose oxidase (1.1.3.5),Catechol oxidase (1.1.3.14), Bilirubin oxidase (1.3.3.5), Alaninedehydrogenase (1.4.1.1), Glutamate dehydrogenase (1.4.1.2), Glutamatedehydrogenase [NAD(P)] (1.4.1.3), Glutamate dehydrogenase (NADP)(1.4.1.4), L-Amino acid dehydrogenase (1.4.1.5), Serine dehydrogenase(1.4.1.7), Valine dehydrogenase (NADP) (1.4.1.8), Leucine dehydrogenase(1.4.1.9), Glycine dehydrogenase (1.4.1.10), L-Amino-acid oxidase(1.4.3.2.), D-Amino-acid oxidase(1.4.3.3), L-Glutamate oxidase(1.4.3.11), Protein-lysine 6-oxidase (1.4.3.13), L-lysine oxidase(1.4.3.14), L-Aspartate oxidase (1.4.3.16), D-amino-acid dehydrogenase(1.4.99.1), Protein disulfide reductase (1.6.4.4), Thioredoxin reductase(1.6.4.5), Protein disulfide reductase (glutathione) (1.8.4.2), Laccase(1.10.3.2), Catalase (1.11.1.6), Peroxidase (1.11.1.7), Lipoxygenase(1.13.11.12), Superoxide dismutase (1.15.1.1).

Said glucose oxidases may be derived from Aspergillus niger.

Said laccases may be derived from Polyporus pinsitus, Myceliophthorathermophila, Coprinus cinereus, Rhizoctonia solani, Rhizoctoniapraticola, Scytalidium thermophilum and Rhus vernicifera. Because of thehomology found between the above mentioned laccases (see WO 98/38287),they are considered to belong to the same class of laccases, namely theclass of “Coprinus-like laccases”. Accordingly, in the present context,the term “Coprinus-like laccase” is intended to indicate a laccasewhich, on the amino acid level, displays a homology of at least 50% andless than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or atleast 55% and less than 100% to the Coprinus cinereus laccase SEQ ID NO:3, or at least 60% and less than 100% to the Coprinus cinereus laccaseSEQ ID NO: 3, or at least 65% and less than 100% to the Coprinuscinereus laccase SEQ ID NO: 3, or at least 70% and less than 100% to theCoprinus cinereus laccase SEQ ID NO: 3, or at least 75% and less than100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 80% andless than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or atleast 85% and less than 100% to the Coprinus cinereus laccase SEQ ID NO:3, or at least 90% and less than 100% to the Coprinus cinereus laccaseSEQ ID NO: 3, at least 95% and less than 100% or at least 98% and lessthan 100% to the Coprinus cinereus laccase SEQ ID NO: 3.

Bilirubin oxidases may be derived from Myrothechecium verrucaria.

The peroxidase may be derived from e.g. Soy bean, Horseradish orCoprinus cinereus.

The protein disulfide reductase may be any of the mentioned in Danishapplication nos. 768/93, 265/94 and 264/94 (Novo Nordisk A/S), which arehereby incorporated as references, including Protein Disulfidereductases of bovine origin, Protein Disulfide reductases derived fromAspergillus oryzae or Aspergillus niger, and DsbA or DsbC derived fromEscherichia coli.

Specific examples of readily available commercial oxidoreductasesinclude Gluzyme™ (enzyme available from Novozymes A/S). However, otheroxidoreductases are available from others.

It is to be understood that also variants of oxidoreductases arecontemplated as the parent enzyme.

The activity of oxidoreductases can be determined as described in“Methods of Enzymatic Analysis”, third edition, 1984, Verlag Chemie,Weinheim, vol. 3.

Parent Carbohydrases

Parent carbohydrases may be defined as all enzymes capable of breakingdown carbohydrate chains (e.g. starches) of especially five and sixmember ring structures (i.e. enzymes classified under the EnzymeClassification number E.C. 3.2 (glycosidases) in accordance with theRecommendations (1992) of the International Union of Biochemistry andMolecular Biology (IUBMB)). Also included in the group of carbohydrasesaccording to the invention are enzymes capable of isomerizingcarbohydrates e.g. six member ring structures, such as D-glucose to e.g.five member ring structures like D-fructose.

Examples include carbohydrases selected from those classified under theEnzyme Classification (E.C.) numbers: alpha-amylase (3.2.1.1),beta-amylase (3.2.1.2), glucan 1,4-alpha-glucosidase (3.2.1.3),cellulase (3.2.1.4), endo-1,3(4)-beta-glucanase (3.2.1.6),endo-1,4-beta-xylanase (3.2.1.8), dextranase (3.2.1.11), chitinase(3.2.1.14), polygalacturonase (3.2.1.15), lysozyme (3.2.1.17),beta-glucosidase (3.2.1.21), alpha-galactosidase (3.2.1.22),beta-galactosidase (3.2.1.23), amylo-1,6-glucosidase (3.2.1.33), xylan1,4-beta-xylosidase (3.2.1.37), glucan endo-1,3-beta-D-glucosidase(3.2.1.39), alpha-dextrin endo-1,6-glucosidase (3.2.1.41), sucrosealpha-glucosidase (3.2.1.48), glucan endo-1,3-alpha-glucosidase(3.2.1.59), glucan 1,4-beta-glucosidase (3.2.1.74), glucanendo-1,6-beta-glucosidase (3.2.1.75), arabinanendo-1,5-alpha-arabinosidase (3.2.1.99), lactase (3.2.1.108),chitonanase (3.2.1.132) and xylose isomerase (5.3.1.5).

Examples of relevant carbohydrases include alpha-1,3-glucanases derivedfrom Trichoderma harzianum; alpha-1,6-glucanases derived from a strainof Paecilomyces; beta-glucanases derived from Bacillus subtilis;beta-glucanases derived from Humicola insolens; beta-glucanases derivedfrom Aspergillus niger, beta-glucanases derived from a strain ofTrichoderma; beta-glucanases derived from a strain of Oerskoviaxanthineolytica; exo-1,4-alpha-D-glucosidases (glucoamylases) derivedfrom Aspergillus niger; alpha-amylases derived from Bacillus subtilis;alpha-amylases derived from Bacillus amyloliquefaciens; alpha-amylasesderived from Bacillus stearothermophilus; alpha-amylases derived fromAspergillus oryzae; alpha-amylases derived from non-pathogenicmicroorganisms; alpha-galactosidases derived from Aspergillus niger,Pentosanases, xylanases, cellobiases, cellulases, hemi-cellulasesderived from Humicola insolens; cellulases derived from Trichodermareesei; cellulases derived from non-pathogenic mold; pectinases,cellulases, arabinases, hemi-celluloses derived from Aspergillus niger,dextranases derived from Penicillium lilacinum; endo-glucanase derivedfrom non-pathogenic mold; pullulanases derived from Bacillusacidopullyticus; beta-galactosidases derived from Kluyveromycesfragilis; xylanases derived from Trichoderma reesei.

Specific examples of readily available commercial carbohydrases includeAlpha-Gal™ Bio-Feed™ Alpha, Bio-Feed™ Beta, Bio-Feed™ Plus, Bio-Feed™Plus, Novozyme® 188, Carezyme® (SEQ ID NO: 5), Celluclast®, Cellusoft®,Ceremyl®, Citrozym™, Denimax™ Dezyme™, Dextrozyme™, Finizym®, Fungamyl™,Gamanase™, Glucanex®, Lactozym®, Maltogenase™, Pentopan™, Pectinex™,Promozyme®, Pulpzyme™, Novamyl™, Termamyl®, AMG (Amyloglucosidase Novo),Maltogenase®, Sweetzyme®, Aquazym®, Natalase® (SEQ ID NO: 4), SP722,AA560 (all enzymes available from Novozymes A/S). Other carbohydrasesare available from other companies.

The parent cellulase is preferably a microbial cellulase. As such, thecellulase may be selected from bacterial cellulases, e.g. Pseudomonascellulases or Bacillus, such as the Bacillus strains described in U.S.Pat. No. 4,822,516, U.S. Pat. No. 5,045,464 or EP 468 464, or B. lautus(cf. WO 91/10732), cellulases. More preferably, the parent cellulasesmay be a fungal cellulase, in particular Humicola, Trichoderma, Irpex,Aspergillus, Penicillium, Myceliophthora or Fusarium cellulases.Examples of suitable parent cellulases are described in, e.g. WO91/17244. Examples of suitable Trichoderma cellulases are thosedescribed in T. T. Teeri, Gene 51, 1987, pp. 43-52. Preferably, theparent cellulase is selected from the cellulases classified in family45, e.g. the enzymes EG B (Pseudomonas fluorescens) and EG V (Humicolainsolens), as described in Henrissat, B. et al.: Biochem. J. (1993),293, p. 781-788.

The Termamyl-Like Alpha-Amylase

It is well known that a number of alpha-amylases produced by Bacillusspp. are highly homologous on the amino acid level. For instance, the B.licheniformis alpha-amylase comprising the amino acid sequence shown inSEQ ID NO: 4 of WO 00/29560 (commercially available as Termamyl®) hasbeen found to be about 89% homologous with the B. amyloliquefaciensalpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 5of WO 00/29560 and about 79% homologous with the B. stearothermophilusalpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 3of WO 00/29560. Further homologous alpha-amylases include analpha-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB12512, NCIB 12513 or DSM 9375, all of which are described in detail inWO 95/26397, and the alpha-amylase described by Tsukamoto et al.,Biochemical and Biophysical Research Communications, 151 (1988), pp.25-31.

Still further homologous alpha-amylases include the alpha-amylaseproduced by the B. licheniformis strain described in EP 0252666 (ATCC27811), and the alpha-amylases identified in WO 91/00353 and WO94/18314. Other commercial Termamyl-like B. licheniformis alpha-amylasesare Optitherm® and Takatherm® (available from Solvay), Maxamyl®(available from Gist-brocades/Genencor), Spezym AA® and Spezyme DeltaAA™ (available from Genencor), and Keistase® (available from Daiwa).

Because of the substantial homology found between these alpha-amylases,they are considered to belong to the same class of alpha-amylases,namely the class of “Termamyl-like alpha-amylases”.

Accordingly, in the present context, the term “Termamyl-likealpha-amylase” is intended to indicate an alpha-amylase which, at theamino acid level, exhibits a substantial homology to Termamyl®, i.e.,the B. licheniformis alpha-amylase having the amino acid sequence shownin SEQ ID NO: 4 (WO 00/29560). In other words, a Termamyl-likealpha-amylase is an alpha-amylase which has the amino acid sequenceshown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or 8 of WO 00/29560, and theamino acid sequence shown in SEQ ID NO: 1 of WO 95/26397 (the same asthe amino acid sequence shown as SEQ ID NO: 7 of WO 00/29560) or in SEQID NO: 2 of WO 95/26397 (the same as the amino acid sequence shown asSEQ ID NO: 8 of WO 00/29560) or in Tsukamoto et al., 1988, (which aminoacid sequence is shown in SEQ ID NO: 6 of WO 00/29560) or i) whichdisplays at least 60% homology (identity), preferred at least 70%, morepreferred at least 75%, even more preferred at least 80%, especially atleast 85%, especially preferred at least 90%, especially at least 95%,even especially more preferred at least 97%, especially at least 99%homology with at least one of said amino acid sequences shown in SEQ IDNOS: 1, 2, 3, 4, 5, 6, 7 or 8 of WO 00/29560 and/or ii) displaysimmunological cross-reactivity with an antibody raised against one ormore of said alpha-amylases, and/or iii) is encoded by a DNA sequencewhich hybridizes, under the low to very high stringency conditions (saidconditions described below) to the DNA sequences encoding theabove-specified alpha-amylases which are apparent from SEQ ID NOS: 9,10, 11, 12, and 32, respectively, of the present application (whichencodes the amino acid sequences shown in SEQ ID NOS: 1, 2, 3, 4, and 5herein, respectively), from SEQ ID NO: 4 of WO 95/26397 (which DNAsequence, together with the stop codon TAA, is shown in SEQ ID NO: 13herein and encodes the amino acid sequence shown in SEQ ID NO: 8 herein)and from SEQ ID NO: 5 of WO 95/26397 (shown in SEQ ID NO: 14 herein),respectively.

In connection with property i), the “homology” (identity) may bedetermined by use of any conventional algorithm, preferably by use ofthe gap progamme from the GCG package version 8 (August 1994) usingdefault values for gap penalties, i.e., a gap creation penalty of 3.0and gap extension penalty of 0.1 (Genetic Computer Group (1991)Programme Manual for the GCG Package, version 8, 575 Science Drive,Madison, Wis., USA 53711).

The parent Termamyl-like alpha-amylase backbone may in an embodimenthave an amino acid sequence which has a degree of identity to SEQ ID NO:4 (WO 00/29560) of at least 65%, preferably at least 70%, preferably atleast 75%, more preferably at least 80%, more preferably at least 85%,even more preferably at least about 90%, even more preferably at least95%, even more preferably at least 97%, and even more preferably atleast 99% identity determined as described above.

A structural alignment between Termamyl® (SEQ ID NO: 4) and aTermamyl-like alpha-amylase may be used to identifyequivalent/corresponding positions in other Termamyl-likealpha-amylases. One method of obtaining said structural alignment is touse the Pile Up programme from the GCG package using default values ofgap penelties, i.e., a gap creation penalty of 3.0 and gap extensionpenalty of 0.1. Other structural alignment methods include thehydrophobic cluster analysis (Gaboriaud et al., (1987), FEBS LETTERS224, pp. 149-155) and reverse threading (Huber, T; Torda, A E, PROTEINSCIENCE Vol. 7, No. 1 pp. 142-149 (1998).

Parent Glucoamylases

Parent glucoamylase contemplated according to the present inventioninclude fungal glucoamylases, in particular fungal glucoamylasesobtainable from an Aspergillus strain, such as an Aspergillus niger orAspergillus awamori glucoamylases and variants or mutants thereof,homologous glucoamylases, and further glucoamylases being structurallyand/or functionally similar to SEQ ID NO: 2 (WO 00/04136). Specificallycontemplated are the Aspergillus niger glucoamylases G1 and G2 disclosedin Boel et al. (1984), “Glucoamylases G1 and G2 from Aspergillus nigerare synthesized from two different but closely related mRNAs”, EMBO J. 3(5), p. 1097-1102. The G2 glucoamylase is disclosed in SEQ ID NO: 2 (WO00/04136). The G1 glucoamylase is disclosed in SEQ ID NO: 13 (WO00/04136). Another AMG backbone contemplated is Talaromyces emersonii,especially Talaromyces emersonii DSM disclosed in WO 99/28448 (NovoNordisk).

The homology referred to above of the parent glucoamylase is determinedas the degree of identity between two protein sequences indicating aderivation of the first sequence from the second. The homology maysuitably be determined by means of computer programs known in the artsuch as GAP provided in the GCG program package (Program Manual for theWisconsin Package, Version 8, August 1994, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch,C. D., (1970), Journal of Molecular Biology, 48, p. 443-453). Using Gapwith the following settings for polypeptide sequence comparison: Gapcreation penalty of 3.0 and Gap extension penalty of 0.1, the maturepart of a polypeptide encoded by an analogous DNA sequence of theinvention exhibits a degree of identity preferably of at least 60%, suchas 70%, at least 80%, at least 90%, more preferably at least 95%, morepreferably at least 97%, and most preferably at least 99% with themature part of the amino acid sequence shown in SEQ ID NO: 2 (WO00/04136).

Preferably, the parent glucoamylase comprise the amino acid sequences ofSEQ ID NO: 2 (WO 00/04136); or allelic variants thereof; or fragmentsthereof that has glucoamylase activity.

A fragment of SEQ ID NO: 2 is a polypeptide which have one or more aminoacids deleted from the amino and/or carboxyl terminus of this amino acidsequence. For instance, the AMG G2 (SEQ ID NO: 2) is a fragment of theAspergillus niger G1 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p.1097-1102) having glucoamylase activity. An allelic variant denotes anyof two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and may result in polymorphism within populations. Gene mutations can besilent (no change in the encoded polypeptide) or may encode polypeptideshaving altered amino acid sequences. An allelic variant of a polypeptideis a polypeptide encoded by an allelic variant of a gene.

It is to be understood that also carbohydrase variants are contemplatedas the parent enzyme.

The activity of carbohydrases can be determined as described in “Methodsof Enzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim,vol. 4.

Parent Transferases

Parent transferases (i.e. enzymes classified under the EnzymeClassification number E.C. 2 in accordance with the Recommendations(1992) of the International Union of Biochemistry and Molecular Biology(IUBMB)) include transferases within this group.

The parent transferases may be any transferase in the subgroups oftransferases: transferases transferring one-carbon groups (E.C. 2.1);transferases transferring aldehyde or residues (E.0 2.2);acyltransferases (E.C. 2.3); glucosyltransferases (E.C. 2.4);transferases transferring alkyl or aryl groups, other that methyl groups(E.C. 2.5); transferases transferring nitrogeneous groups (2.6).

In a preferred embodiment the parent transferase is a transglutaminaseE.C 2.3.2.13 (Protein-glutamine μ-glutamyltransferase).

Transglutaminases are enzymes capable of catalyzing an acyl transferreaction in which a gamma-carboxyamide group of a peptide-boundglutamine residue is the acyl donor. Primary amino groups in a varietyof compounds may function as acyl acceptors with the subsequentformation of monosubstituted gamma-amides of peptide-bound glutamicacid. When the epsilon-amino group of a lysine residue in apeptide-chain serves as the acyl acceptor, the transferases formintramolecular or intermolecular gamma-glutamyl-epsilon-lysylcrosslinks.

Examples of transglutaminases are described in the pending DK patentapplication no. 990/94 (Novo Nordisk A/S).

The parent transglutaminase may be of human, animal (e.g. bovine) ormicrobial origin.

Examples of such parent transglutaminases are animal derivedTransglutaminase, FXIIIa; microbial transglutaminases derived fromPhysarum polycephalum (Klein et al., Journal of Bacteriology, Vol. 174,p. 2599-2605); transglutaminases derived from Streptomyces sp.,including Streptomyces lavendulae, Streptomyces lydicus (formerStreptomyces libani) and Streptoverticillium sp., includingStreptoverticillium mobaraense, Streptoverticillium cinnamoneum, andStreptoverticillium griseocarneum (Motoki et al., U.S. Pat. No.5,156,956; Andou et al., U.S. Pat. No. 5,252,469; Kaempfer et al.,Journal of General Microbiology, Vol. 137, p. 1831-1892; Ochi et al.,International Journal of Sytematic Bacteriology, Vol. 44, p. 285-292;Andou et al., U.S. Pat. No. 5,252,469; Williams et al., Journal ofGeneral Microbiology, Vol. 129, p. 1743-1813).

It is to be understood that also transferase variants are contemplatedas the parent enzyme.

The activity of transglutaminases can be determined as described in“Methods of Enzymatic Analysis”, third edition, 1984, Verlag Chemie,Weinheim, vol. 1-10.

Parent Phytases

Parent phytases are included in the group of enzymes classified underthe Enzyme Classification number E.C. 3.1.3 (Phosphoric MonoesterHydrolases) in accordance with the Recommendations (1992) of theInternational Union of Biochemistry and Molecular Biology (IUBMB)).

Phytases are enzymes produced by microorganisms which catalyse theconversion of phytate to inositol and inorganic phosphorus

Phytase producing microorganisms comprise bacteria such as Bacillussubtilis, Bacillus natto and Pseudomonas; yeasts such as Saccharomycescerevisiae; and fungi such as Aspergillus niger, Aspergillus ficuum,Aspergillus awamori, Aspergillus oryzae, Aspergillus terreus orAspergillus nidulans, and various other Aspergillus species).

Examples of parent phytases include phytases selected from thoseclassified under the Enzyme Classification (E.C.) numbers: 3-phytase(3.1.3.8) and 6-phytase (3.1.3.26).

The activity of phytases can be determined as described in “Methods ofEnzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim, vol.1-10, or may be measured according to the method described in EP-A1-0420 358, Example 2A.

Lyases

Suitable lyases include Polysaccharide lyases: Pectate lyases (4.2.2.2)and pectin lyases (4.2.2.10), such as those from Bacillus licheniformisdisclosed in WO 99/27083.

Isomerases Protein Disulfide Isomerase

Without being limited thereto suitable protein disulfide isomerasesinclude PDIs described in WO 95/01425 (Novo Nordisk A/S) and suitableglucose isomerases include those described in Biotechnology Letter, Vol.20, No 6, June 1998, pp. 553-56.

Contemplated isomerases include xylose/glucose Isomerase (5.3.1.5)including Sweetzyme®.

Environmental Allergens

The environmental allergens that are of interest for epitope mappinginclude allergens from pollen, dust mites, mammals, venoms, fungi, fooditems, and other plants.

Pollen, allergens include but are not limited to those of the orderFagales, Oleales, Pinales, Poales, Asterales, and Urticales; includingthose from Betula, Alnus, Corylus, Carpinus, Olea, Phleum pratense andArtemisia vulgaris, such as Aln g1, Cor a1, Car b1, Cry j1, Amb a1 anda2, Art v1, Par j1, Ole e1, Ave v1, and Bet v1 (WO 99/47680).

Mite allergens include but are not limited to those from Derm. farinaeand Derm. pteronys., such as Der f1 and f2, and Der p1 and p2.

From mammals, relevant environmental allergens include but are notlimited to those from cat, dog, and horse as well as from dandruff fromthe hair of those animals, such as Fel d1; Can f1; Equ c1; Equ c2; Equc3.

Venum allergens include but are not limited to PLA2 from bee venom aswell as Apis m1 and m2, Ves g1, g2 and g5, Ves v5 and to Pol and Solallergens.

Fungal allergens include those from Alternaria alt. and Cladospo. herb.such as Alt a1 and Cla h1.

Food allergens include but are not limited to those from milk(lactoglobulin), egg (ovalbumin), peanuts, hazelnuts, wheat(alpha-amylase inhibitor),

Other plant allergens include latex (hevea brasiliensis).

In addition, a number of proteins of interest for expression intransgenic plants could be useful objects for epitope engineering. Iffor instance a heterologous enzyme is introduced into a transgenic plante.g. to increase the nutritional value of food or feed derived from thatplant, that enzyme may lead to allergenicity problems in humans oranimals ingesting the plant-derived material. Epitope mapping andengineering of such heterologous enzymes or other proteins of transgenicplants may lead to reduction or elimination of this problem. Hence, themethods of this patent are also useful for potentially modifyingproteins for heterologous expression in plants and plant cells.

Materials and Methods Materials ELISA Reagents:

Horse Radish Peroxidase labelled pig anti-rabbit-Ig (Dako, DK, P217,dilution 1:1000)Rat anti-mouse IgE (Serotec MCA419; dilution 1:100)Mouse anti-rat IgE (Serotec MCA193; dilution 1:200)Biotin-labelled mouse anti-rat IgG1 monoclonal antibody (Zymed 03-9140;dilution 1:1000)Biotin-labelled rat anti-mouse IgG1 monoclonal antibody (SerotecMCA336B; dilution 1:2000)Streptavidin-horse radish peroxidase (Kirkegård & Perry 14-30-00;dilution 1:1000).

Buffers and Solutions:

PBS (pH 7.2 (1 liter))

NaCl 8.00 g KCl 0.20 g K₂HPO₄ 1.04 g KH₂PO₄ 0.32 g

Washing buffer PBS, 0.05% (v/v) Tween 20

Blocking buffer PBS, 2% (wt/v) Skim Milk powder

Dilution buffer PBS, 0.05% (v/v) Tween 20, 0.5% (wt/v) Skim Milk powder

Citrate buffer 0.1M, pH 5.0-5.2

Stop-solution (DMG-buffer)

Sodium Borate, borax (Sigma)

3,3-Dimethyl glutaric acid (Sigma)

Tween 20: Poly oxyethylene sorbitan mono laurate (Merck cat no. 822184)

PMSF (phenyl methyl sulfonyl flouride) from Sigma

Succinyl-Alanine-Alanine-Proline-Phenylalanine-paranitro-anilide(Suc-AAPF-pNP) Sigma no. S-7388, Mw 624.6 g/mol.

mPEG (Fluka)

Coloring Substrate:

OPD: o-phenylene-diamine, (Kementec cat no. 4260)

Methods Automatic Epitope Mapping Implementation

The implementation consists of 3 pieces of code:

1. The core program (see above), written in C (see Appendix A).2. A “wrapping” cgi-script run by the web server, written in Python (seeAppendix B).3. A HTML page defining the input/submission form (see Appendix C).

The wrapper receives the input and calls the core program and severalother utilities.

Apart from the standard Unix utility programs (my, rm, awk, etc.) thefollowing must be installed:

A web server capable of running cgi-scripts, e.g. Apache

Python 1.5 or later

Gnuplot 3.7 or later

DSSP, version July 1995

The Core Program Inputs

1. A Brookhaven PDB file with the structure of the protein2. The output of DSSP called with the above PDB file.3. Maximum distance between adjacent residues4. Minimum solvent accessible surface area for each residue5. Maximum epitope size (max distance between any two residues inepitope)6. Maximum number of non-redundant epitopes to include (0=all)7. The shortest acceptable epitope (as a fraction of the length of theepitope consensus sequence).8. Epitope consensus sequence describing which residues are possible atthe different positions. An example is shown below:KR (Lys or Arg allowed)AILV− (Ala, Ile, Leu, Val or missing residue allowed)* (All Residues Allowed, but there Must be a Residue)

? (All or Missing Residue Allowed)

DE (Asp or Glu allowed)(*, ? or − in first or last position is allowed but obsolete. (− infirst position is ignored.))

Examples of matching epitopes:

KAAKD (SEQ ID NO: 41), KLASD (SEQ ID NO: 42), KLYSD (SEQ ID NO: 43),KLY-D (SEQ ID NO: 44), R-M-D. The Epitope Searching Algorithm

The “core” of the program is the algorithm that scans the proteinsurface for the epitope patterns. The principle is that several “trees”are built, where each of their branches describes one epitope:

1. All residues in the protein are checked according to: a) Does theresidue type match the first residue of the epitope consensus sequence.b) Is the surface accessibility greater than or equal to the giventhreshold. If both requirements are fulfilled, the protein residue isconsidered as one root in the epitope tree. Remark that there areusually many roots.2. For each of the residues defined as roots, all residues within thegiven threshold distance between adjacent residues (e.g. 7 Angstroms)are checked for the same as above: a) Does the residue type match thesecond residue of the epitope consensus sequence. b) Is the surfaceaccessibility greater than or equal to the given threshold. If yes, theprotein residue is considered as a “child” of the root. The spatialposition of a residue is defined as the coordinates of its C-alpha atom.3. The procedure from step 2 is repeated for the next residue in theepitope consensus sequence, where each of the “childs” found in step 2are now “roots” of new childs. If a gap is defined in the epitopeconsensus sequence, a “missing” residue is allowed, and the coordinatesof the root (also called “parent”) is used.4. This procedure is repeated for all residues in the epitope consensussequence.5. In this way a number of trees (corresponding to the number of rootsfound in step 1) are found. Notice that the same protein residue can bepresent many places in the trees.6. If no epitopes that matches the length of the epitope consensussequence are found, the longest shorter epitopes that matches the firstn residues of the epitope consensus sequence are used, where n is aninteger smaller than the length of the epitope consensus sequence. If nis smaller than the length of the epitope consensus sequence multipliedby the fraction value defining the shortest acceptable epitope length,no epitopes are written to the output, and steps 7, 8 and 9 are skipped.7. The epitopes are extracted from the trees by traversing down fromeach of the “childs” in the last level. The algorithm also findsepitopes which have the same protein residue present more than once.This is, of course, an artifact and such epitopes are discarded. Everyepitope is then checked for its size, that is, the maximum distancebetween any two residues which are members of the epitope. If thisexceeds the threshold, the epitope is discarded.8. Redundant epitopes are removed. Epitopes containing one or more gapsare redundant if they are subsets of other epitopes without or withfewer gaps. For example: A82-gap-F45-G44-K43 is a subset ofA82-L46-F45-G44-K43, and is therefore discarded.9. For every epitope, the total solvent accessible surface area iscalculated (by adding the contributions from each residue as found bythe DSSP program). The epitopes are sorted according to this area indescending order. If a maximum number of n non-redundant epitopes hasbeen specified, the n epitopes with largest solvent accessible surfacearea are selected.10. The output consists of a list of the found epitopes, along withinformation of the epitope consensus sequence used and other internalparameters. A separate file containing the number of epitopes that eachof the protein residues is a member of is also written.The wrapper

Inputs

1. One PDB file, describing one structure, or one ZIP file, containing anumber of PDB files, each describing one structure. The ZIP file mustnot contain subfolders.2. An epitope consensus sequence or which part of the current epitopelibrary to use (full library or IgE part or IgG part).3. Maximum distance between adjacent residues4. Minimum solvent accessible surface area for each residue5. Maximum epitope size (max distance between any two residues inepitope)6. Maximum number of non-redundant epitopes to include (0=all)7. Whether to use sequential numbering (1,2,3,4, etc) or PDB-filenumbering.

DESCRIPTION

The core program accepts only one structure and one epitope consensussequence. It is usually desirable to use a library of epitope consensussequences and sometimes several protein structures. The wrapper readsthe user input and calls the utility programs and the core program thenecessary number of times. The output is collected and presented on theweb page returned to the user.

Depending on the type of input, the wrapper works in different modes:

Epitope consensus can be given directly or taken from a library

Input type can be a single PDB file or a collection of PDB file given asa ZIP-file.

Any of the four possible combinations are allowed.

The epitope library consists of a number of text files, each containingone epitope consensus sequence as specified above.

The layout of the wrapper is like this:

1. Check if the program is already in use from somewhere else (this isdone by checking for a lock file when the wrapper starts. If it does notexist, it is created and removed again when the program is finished).2. If the epitope consensus sequences are to be read from the library,make an internal list of the desired library entries.3. If the input type is a ZIP file, unzip the file and create one newdirectory for each of the contained PDB files. Move each PDB file to itscorresponding directory.4. Do a loop over the structures and/or epitope consensus sequences. Foreach structure/epitope consensus sequence pair, DSSP and the coreprogram is called with the required parameters. If the input type is aZIP file, the outputs are put in the appropriate directories.5. If the epitope library is used, a sum file containing the totalnumber of epitopes each residue is a member of. (Such a file isgenerated by the core program for each epitope consensus sequence—here asum of these files is calculated). If input type is a ZIP file, a sumfile is generated for each structure and put in the appropriatedirectory.6. If the epitope library is used, a file containing the total number ofepitopes found from each entry in the epitope library. If the input typeis a PDB file, the file contains only one line (with a number of datacorresponding to the library size). If the input type is a ZIP file,there is one line for each structure.7. Depending on the combination of input type (ZIP or single PDB) andepitope consensus sequence source (typed-in or epitope library),different information is returned to the user:Single PDB+typed in epitope: Graph of numbers of epitopes that eachresidue is a member of. List of found epitopes.ZIP file+typed in epitope: Graphs (one for each structure) of numbers ofepitopes that each residue is a member of. Lists (one for eachstructure) of found epitopes.Single PDB+epitope library: Graph of numbers of epitopes that eachresidue is a member of (total for the complete library).ZIP file+epitope library: Graphs (one for each structure) of numbers ofepitopes that each residue is a member of (total for the completelibrary).Data flow sheets for the four different are shown in the FIG.8. For all modes except Single PDB+typed in epitope, a ZIP filecontaining all output files is created and returned to the user.

Immunisation of Brown Norway Rats:

Twenty intratracheal (IT) immunisations were performed weekly with 0,100ml 0.9% (wt/vol) NaCl (control group), or 0,100 ml of a protein dilution(˜0, 1-1 mg/ml). Each group contained 10 rats. Blood samples (2 ml) werecollected from the eye one week after every second immunisation. Serumwas obtained by blood clothing and centrifugation and analysed asindicated below.

Immunisation of Balb/C Mice:

Twenty subcutaneous (SC) immunisations were performed weekly with 0.05ml 0.9% (wt/vol) NaCl (control group), or 0.050 ml of a protein dilution(˜0.01-0.1 mg/ml). Each group contained 10 female Balb/C mice (about 20grams) purchased from Bomholdtgaard, Ry, Denmark. Blood samples (0.100ml) were collected from the eye one week after every secondimmunisation. Serum was obtained by blood clothing and centrifugationand analysed as indicated below.

ELISA Procedure for Detecting Serum Levels of IgE and IgG:

Specific IgG1 and IgE levels were determined using the ELISA specificfor mouse or rat IgG1 or IgE. Differences between data sets wereanalysed by using appropriate statistical methods.

Activation of CovaLink Plates:

A fresh stock solution of cyanuric chloride in acetone (10 mg/ml) isdiluted into PBS, while stirring, to a final concentration of 1 mg/mland immediately aliquoted into CovaLink NH2 plates (100 microliter perwell) and incubated for 5 minutes at room temperature. After threewashes with PBS, the plates are dryed at 50° C. for 30 minutes, sealedwith sealing tape, and stored in plastic bags at room temperature for upto 3 weeks.

Mouse anti-Rat IgE was diluted 200× in PBS (5 microgram/ml). 100microliter was added to each well. The plates were coated overnight at4° C.

Unspecific adsorption was blocked by incubating each well for 1 hour atroom temperature with 200 microliter blocking buffer. The plates werewashed 3× with 300 microliter washing buffer.

Unknown rat sera and a known rat IgE solution were diluted in dilutionbuffer: Typically 10×, 20× and 40× for the unknown sera, and 1/2dilutions for the standard IgE starting from 1 μg/ml. 100 microliter wasadded to each well. Incubation was for 1 hour at room temperature.

Unbound material was removed by washing 3× with washing buffer. Theanti-rat IgE (biotin) was diluted 2000× in dilution buffer. 100microliter was added to each well. Incubation was for 1 hour at roomtemperature. Unbound material was removed by washing 3× with washingbuffer.

Streptavidin was diluted 1000× in dilution buffer. 100 microliter wasadded to each well. Incubation was for 1 hour at room temperature.Unbound material was removed by washing 3× with 300 microliter washingbuffer. OPD (0.6 mg/ml) and H₂O₂ (0.4 microliter/ml) were dissolved incitrate buffer. 100 microliter was added to each well. Incubation wasfor 30 minutes at room temperature. The reaction was stopped by additionof 100 microliter H₂SO₄. The plates were read at 492 nm with 620 nm asreference.

Similar determination of IgG can be performed using anti Rat-IgG andstandard rat IgG reagents.

Similar determinations of IgG and IgE in mouse serum can be performedusing the corresponding species-specific reagents.

Direct IgE Assay:

To determine the IgE binding capacity of protein variants one can use anassay, essentially as described above, but using sequential addition ofthe following reagents:

1) Mouse anti-rat IgE antibodies coated in wells;2) Known amounts of rat antiserum containing IgE against the parentprotein;3) Dilution series of the protein variant in question (or parent proteinas positive control);4) Rabbit anti-parent antibodies5) HRPO-labelled anti-rabbit Ig antibodies for detection using OPD asdescribed.

The relative IgE binding capacity (end-point and/or affinity) of theprotein variants relative to that of the parent protein are determinedfrom the dilution-response curves. The IgE-positive serum can be ofother animals (including humans that inadvertently have been sensitizedto the parent protein) provided that the species-specific anti-IgEcapture antibodies are changed accordingly.

Competitive ELISA (C-ELISA):

C-ELISA was performed according to established procedures. In short, a96 well ELISA plate was coated with the parent protein. After properblocking and washing, the coated antigen was incubated with rabbitanti-enzyme polyclonal antiserum in the presence of various amounts ofmodified protein (the competitior). The residual amount of rabbitantiserum was detected by horseraddish peroxidase-labelled piganti-rabbit immunoglobulin.

Protein Sequences and Alignments:

For purposes of the present invention, the degree of homology may besuitably determined by means of computer programs known in the art, suchas GAP provided in the GCG program package (Program Manual for theWisconsin Package, Version 8, August 1994, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch,C. D., (1970), Journal of Molecular Biology, 48, 443-45).

Subtilisin Proteases:

In the present invention, corresponding (or homologous) positions insubtilisin protease sequences are defined by alignment with SubtilisinNovo (BPN′) from B. amyloliquefaciens, as shown in Table 1A forAlcalase, Protease B, Esperase, Protease C, Protease D, Protease E,Protease A, PD498, Properase, Relase, Savinase.

TABLE 1A Alignment of different proteases to the sequence of BPN′Alcalase: 69.5% identity in 275 residues overlap; Score: 953.0; Gapfrequency: 0.4% Alcalase, 1AQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYN-TD BPN′, 1AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ******   ***     **  * ****** * **  ***** * **** *  *  *  * Alcalase, 60GNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMD BPN′, 61DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  ************* *  ********  *******   *** ** *  *****  * ** Alcalase, 120VINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAV BPN′, 121VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV *********** * * *** * * ********** ** *   * *** ** ******** Alcalase, 180DSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPN BPN′, 181DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN**   ******** ** *****    ** * * *   *********************** Alcalase,240 LSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ (SEQ ID NO: 45) BPN′, 241WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46)     ***  *  * * ************* **** Protease B: 59.6% identity in 275 residues overlap;Score: 820.0; Gap frequency: 2.2% PROTEASE B, 1AQTIPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD **  * **   *** *  * *** ***** * ** * ****   **** ** * *  ** PROTEASE B, 59GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMH BPN′, 61DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ***********************  ********  ***  * *  * ***  * * PROTEASE B, 119VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT BPN′, 121VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** **** *  **  *   ** **** ** *         **  *    **** PROTEASE B, 175DQNNNRASFSQYGAGLDIMAPGVNIQSTYPGSTYASDNGTSMATPHVAGAAALVKQKNPS BPN′, 181DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  ******  *  ** ***** **** **  *   ****** *********   * * PROTEASE B, 235WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 47) BPN′, 241WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** ***    ** ** *  ** Esperase: 54.7% identity in 274 residues overlap;Score: 745.0; Gap frequency: 2.2% Esperase, 1QTVPWGISFINTQQAHNRGIFGNGARVAVLDTGI-ASHPDLRIAGGASFISSE-PSYHDN BPN′, 2QSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQDD * ** * **     *  *  *    *** * **  *****  *****   ** *   * Esperase, 59NGHGTHVAGTIAALNNSIGVLGVAPSADLYAVKVLDRNGSGSLASVAQGIEWAINNNMHI BPN′, 62NSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMDV * ***********************  *******   ***       ****** *** Esperase, 119INMSLGSTSGSSTLELAVNRANNAGILLVGAAGNTGRQG----VNYPARYSGVMAVAAVD BPN′, 122INMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAVD******  ***  *  **  *   *   * **** *  *    * **  *  * ** *** Esperase,175 QNGQRASFSTYGPEIEISAPGVNVNSTYTGNRYVSLSGTSMATPHVAGVAALVKSRYPSY BPN′,182 SSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNW   ******  ***    ****   **  ** *    ***** ***** ***  *  * Esperase, 235TNNQIRQRINQTATYLGSPSLYGNGLVHAGRATQ (SEQ ID NO: 48) BPN′, 242TNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) ** * *     * ***    ** **     * * Protease C: 59.6% identity in 275 residues overlap;Score: 825.0; Gap frequency: 2.2% ProteaseC, 1AQSVPWGISRVQAPAAHNRGLTGSGVRVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** **   *** *  * *** * *** * ** * ****   **** ** * *  ** ProteaseC, 59GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSYSSIAQGLEWAGNNGMH BPN′, 61DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ***********************  ********  *** ** *  * ***  * * ProteaseC, 119VASLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT BPN′, 121VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV *   *** **** *  **  *   ** **** ** *         **  *    **** ProteaseC, 175DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS BPN′, 181DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  ******  *  **  ****  *** **  *   ****** *********   * * ProteaseC, 235WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAAAR (SEQ ID NO: 49) BPN′, 241WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** ***    ** ** *  *** Protease D: 59.3% identity in 275 residues overlap;Score: 815.0; Gap frequency: 2.2% ProteaseD, 1AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** **   *** *  * *** ***** * ** * ****   **** ** * *  ** ProteaseD, 59GNGHGTHVAGTIAALDNSIGVLGVAPSAELYAVKVLGASGSGAISSIAQGLEWAGNNGMH BPN′, 61DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * *********** ***********  ********  ***  * *  * ***  * * ProteaseD, 119VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT BPN′, 121VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** **** *  **  *   ** **** ** *         **  *    **** ProteaseD, 175DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS BPN′, 181DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  ******  *  **  ****  *** **  *   ****** *********   * * ProteaseD, 235WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 50) BPN′, 241WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** ***    ** ** *  ** Protease E: 58.2% identity in 275 residues overlap;Score: 800.0; Gap frequency: 2.2% ProteaseE, 1AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** **   *** *  * *** ***** * ** * ****   **** ** * *  ** ProteaseE, 59GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGGGAISSIAQGLEWAGNNGMH BPN′, 61DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ***********************  ********  * *  * *  * ***  * * ProteaseE, 119VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----DSISYPARYANAMAVGAT BPN′, 121VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** **** *  **  *   ** **** ** *         **  *    **** ProteaseE, 175DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAVLVKHKNPS BPN′, 181DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  ******  *  **  ****  *** **  *   ****** ******* *   * * ProteaseE, 235WSNVRIRDHLKKTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 51) BPN′, 241WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * *   *  *  * ***    ** ** *  ** Protease A: 58.9% identity in 275 residues overlap;Score: 812.0; Gap frequency: 2.2% Protease A, 1AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** **   *** *  * *** ***** * ** * ****   **** ** * *  ** Protease A, 59GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMH BPN′, 61DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ***********************  ********  ***  * *  * ***  * * Protease A, 119VANLSLGSPSAGGTLEQAVNSATSRGVLVVAASGNSGA----GSISAPASYANAMAVGAT BPN′, 121VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *****    *  **  *   ** **** ** *          *  *    **** Protease A, 175DQNNNRASFSQYGPGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS BPN′, 181DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  ******  ** **  ****  *** ** *   ****** *********   * * Protease A, 235WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 52) BPN′, 241WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** ***    ** ** *  ** PD498: 47.7% identity in 266 residues overlap; Score:487.0; Gap frequency: 4.9% PD498, 13YGPQNTSTPAAWDVTRGSSTQTVAVLDSGVDYNHPDLARKVIKGYDFIDRDN-NPMDLNG BPN′, 6YGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDL--KVAGGASMVPSETPNFQDDNS**      **        *   *** *** *  ****  **  *         *  * * PD498, 72HGTHVAGTVAADTNNGIGVAGMAPDTKILAVRVLDANGSGSLDSIASGIRYAADQGAKVL BPN′, 64HGTHVAGTVAA-LNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMDVI***********  ** *** * **     ** **   ***    *  **  *      * PD498, 132NLSLGCECNSTTLKSAVDYAWNKGAVVVAAAGND----NVSRTFQPASYPNAIAVGAIDS BPN′, 123NMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAVDS ****    *  ** *** *   * ********       *    *  **  ***** ** PD498, 188NDRKASFSNYGTWVDVTAPGVNIASTVPNNGYSYMSGTSMASPHVAGLAALLASQGKN-- BPN′, 183SNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWT    ****  *   ** **** * ** * * *    *********** ***  *   * PD498, 246NVQIRQAIEQTADKISGTGTNFKYGK (SEQ ID NO: 53) BPN′, 243NTQVRSSLQNTTTKL---GDSFYYGK (SEQ ID NO: 46) * * *     *  *    *  * ***Properase: 58.9% identity in 275 residues overlap; Score: 813.0; Gapfrequency: 2.2% Properase, 1AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** **   *** *  * *** ***** * ** * ****   **** ** * *  ** Properase, 59GNGHGTHVAGTIAALNNSIGVLGVAPNAELYAVKVLGASGGGSNSSIAQGLEWAGNNGMH BPN′, 61DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * **********************   ********  * *  * *  * ***  * * Properase, 119VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT BPN′, 121VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** **** *  **  *   ** **** ** *         **  *    **** Properase, 175DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS BPN′, 181DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  ******  *  **  ****  *** **  *   ****** *********   * * Properase, 235WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 54) BPN′, 241WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** ***    ** ** *  ** Relase: 60.7% identity in 275 residues overlap; Score:858.0; Gap frequency: 1.8% Relase, 1AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGIDSTHPDLNIRGGASFVPGE-PSTQD BPN′, 1AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** **   *** *  * *** ***** * **** ****   **** ** * *  ** Relase, 60GNGHGTHVAGTIAALDNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMD BPN′, 61DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * *********** ***********  ********  ***  * *  * ***  * ** Relase, 120VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT BPN′, 121VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** **** *  **  *   ** **** ** *         **  *    **** Relase, 176DQNNNRASFSQYGAELDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVLQKNPS BPN′, 181DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  ******  * ***  ****  *** **  *   ****** ********* * * * Relase, 236WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 55) BPN′, 241WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** ***    ** ** *  ** Savinase: 59.6% identity in 275 residues overlap;Score: 821.0; Gap frequency: 2.2% Savinase, 1AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** **   *** *  * *** ***** * ** * ****   **** ** * *  ** Savinase, 59GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMH BPN′, 61DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ***********************  ********  ***  * *  * ***  * * Savinase, 119VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT BPN′, 121VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** **** *  **  *   ** **** ** *         **  *    **** Savinase, 175DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS BPN′, 181DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  ******  *  **  ****  *** **  *   ****** *********   * * Savinase, 235WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 56) BPN′, 241WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** ***    ** ** *  **

To find the homologous positions in subtilisin protease sequences notshown in the alignment of Table 1A, the sequence of interest is alignedto the sequence of BPN′ as shown in Table 1B for YaB protease andSubtilisin sendai. The new sequence is aligned to the BPN′ sequence byusing the GAP alignment to the most homologous sequence found by the GAPprogram. GAP is provided in the GCG program package (Program Manual forthe Wisconsin Package, Version 8, August 1994, Genetics Computer Group,575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. andWunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45).

The sequence of the YaB protease is disclosed by Kaneko, R.; Koyama, N.;Tsai, Y.-C.; Juang, R.-Y.; Yoda, K.; Yamasaki, M.; Molecular cloning ofthe structural gene for alkaline elastase YaB, a new subtilisin producedby an alkalophilic Bacillus strain. J. Bacteriol. 171:5232 (1989), ithas Swissprot number P20724, and is shown in SEQ ID NO: 35.

The sequence of the Subtilisin sendai is disclosed by Yamagata, Y.;Isshiki, K.; Ichishima, E.; Subtilisin Sendai from alkalophilic Bacillussp.: molecular and enzymatic properties of the enzyme and molecularcloning and characterization of the gene, aprS. Enzyme Microb. Technol.17:653 (1995), it has SPTREMBL accession number Q45522, and is shown inSEQ ID NO: 34.

Identity to savinase: 81.7%identity to savinase: 82.09%

Swissprot: P20724

TABLE 1B Alignment of YAB protease to BPN′: 55.3% identity CLUSTAL W(1.7) multiple sequence alignment YAB-QTVPWGINRVQAPIAQSRGFTGTGVRVAVLDTGISN-HADLRIRGGASFVPGE-PNISD BPN{acuteover ( )} AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD *:**:*:.:::**  :*:*:**:.*:***:*:**.. *.**:: ****:**.* **:.* YABGNGHGTQVAGTIAALNNSIGVLGVAPNVDLYGVKVLGASGSGSISGIAQGLQWAANNGMH BPN{acuteover ( )} DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD.*.***:****:**************.  **.***** :***. * * :*::**  *.*. YABIANMSLGSSAGSATMEQAVNQATASGVLVVAASGNSG----AGNVGFPARYANAMAVGAT BPN{acuteover ( )} VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV :*****..:***::: **::*.****:****:**.*    :..**:*.:*...:****. YABDQNNNRATFSQYGAGLDIVAPGVGVQSTVPGNGYASFNGTSMATPHVAGVAALVKQKNPS BPN{acuteover ( )} DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN*..*:**:**. *. **::****.:***:*** *.::******:*****.***: .*:*. YABWSNVQIRNHLKNTATNLGNTTQFGSGLVNAEAATR (SEQ ID NO: 57) BPN{acute over ( )}WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) *:*.*:*.*:**:*:**::  :*.**:*.:**:: Alignment of Subtilisin sendai to BPN′: 55.6%identity. CLUSTAL W (1.7) multiple sequence alignment sendaiNQVTPWGITRVQAPTAWTRGYTGTGVRVAVLDTGIS-THPDLNIRGGVSFVPGE-PSYQD BPN{acuteover ( )} AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD * .*:*:::::**:  ::****:.*:***:*:**. :****:: **.*:**.* *.:** sendaiGNGHGTHVAGTIAALNNSIGVVGVAPNAELYAVKVLGANGSGSVSSIAQGLQWTAQNNIH BPN{acuteover ( )} DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD.*.********:*********:****.: ********  ***. * * :*::*:  **:. sendaiVANLSLGSPVGSQTLELAVNQATNAGVLVVAATGNNG----SGTVSYPARYANALAVGAT BPN{acuteover ( )} VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV **:***.* ** :*: **::*. :**:****:**:*    *.**.**.:*...:****. sendaiDQNNNRASFSQYGTGLNIVAPGVGIQSTYPGNRYASLSGTSMATPHVAGVAALVKQKNPS BPN{acuteover ( )} DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN*..*:*****. *. *:::****.**** ***:*.: .*****:*****.***: .*:*. sendaiWSNTQIRQHLTSTATSLGNSNQFGSGLVNAEAATR (SEQ ID NO: 58) BPN{acute over ( )}WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) *:***:*. *.*:*.**:*  :*.**:*.:**::

These alignments reveal that that homology between various subtilisinproteases ranges between 100% and 40%.

Unless specified, subtilisin sequences and positions mentioned in thepresent invention, are given in the BPN′ numeration, and can beconverted by alignment as 50 described above (Tables 1A and 1B).

Sequence identities between different pairs of proteases are givenbelow:

Sequence identity to BPN′:

Savinase 60.4% Alcalase 69.5% BLAPR 60.4% ProteaseC 0.4% ProteaseD 0.0%ProteaseE 8.2% Protease A 0.0% Properase 9.6% Relase 61.5% PD498 44.8%sendai 55.6% YAB 55.3%Sequence identity to Savinase:

Alcalase 60.9% BLAPR 98.1% ProteaseC 8.5% ProteaseD 8.9% ProteaseE 6.7%Protease A 7.8% Properase 8.9% Relase 98.1% PD498 44.3% sendai 81.4% YAB81.8%

Structures

The protein structure of PD498 is disclosed in WO 98/35026 (NovoNordisk). The structure of Savinase can be found in BETZEL et al,J.MOL.BIOL., Vol. 223, p. 427, 1992 (1 svn.pdb).

Homology Modelling

Three dimensional structural models of the subtilisins properase,release, ProteaseC, ProteaseD, ProteaseE, and PROTEASE B wereconstructed based on three dimensional structure of Savinase (ProteinData Bank entry 1SVN; Betzel, C., Klupsch, S., Papendorf, G., Hastrup,S., Branner, S., Wilson, K. S.: Crystal structure of the alkalineproteinase Savinase from Bacillus lentus at 1.4 Å resolution. J Mol Biol223 pp. 427 (1992)) using the Modeller 5o ( Sali, A.; T. L. Blundell,“Definition of general topological equivalence in protein structures: Aprocedure involving comparison of properties and relationships throughsimulated annealing and dynamic programming,” J. Mol. Biol., 212 403-428(1990)) module of the Insight 2000 molecular modelling package (Biosyminc.). Default parameters were used with the alignments shown in FIG. 1Aas input, e.g. alignment between the columns labelled Savinase andPROTEASE B served as input alignment in construction of a PROTEASE Bstructural model. The Modeller module by default output ten structuralmodels, of these the model with lowest ‘modeller objective function’score was chosen as representing PROTEASE B structure.

Lipase:

The sequence of the T. lanuginosus lipase (trade name Lipolase) isprovided in SEQ ID NO: 1 and the structure is disclosed in WO 98/35026and as “1tib”, available in Structural Classification of Proteins (SCOP)on the Internet.

Amylase:

The amylase used in the examples is the alpha-amylase of Bacillushalmapalus (WO 96/23873), which is called amylase SP722 (the wild-type).Its sequence is shown in SEQ ID NO: 2 and the corresponding proteinstructure was built from the BA2 structure, as described in WO 96/23874.The first four amino acids of the structural model are not defined,hence the sequence used for numeration of amino acid residues in theexamples of this invention is four amino acids shorter than the one ofthe full length protein SP722.

Several variants of this amylase are available (WO 96/23873). Oneparticularly useful variant has deleted two amino acid residues at D-Gat positions 183 and 184 of the SEQ ID NO: 2 (corresponding to residues179 and 180 of the modelled structure). This variant is called JE-1 orNatalase.

Another amylase that is particularly useful is the amylase AA560: Thisalkaline alpha-amylase may be derived from a strain of Bacillus sp. DSM12649. The strain was deposited on 25 Jan. 1999 by the assignee underthe terms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure atDeutshe Sammmlung von Microorganismen and Zellkulturen GmbH (DSMZ),Mascheroder Weg 1b, D-38124 Braunschweig DE.

Laccase:

The laccase used in this invention is that from Coprinus cinereus (WO98/38287), the sequence of which is shown as SEQ ID NO: 3. The structureof the Myceliophthora thermophila laccase can be built by homologymodeling to the Coprinus cinereus laccase as shown in WO 98/38287.

Cellulase:

The cellulase sequence and structure used in the present invention isthat of the core fragment of endoglucanase V from Humicola insolens (akaCel45 or Carezyme). The core fragment structure is available as 3eng.pdb(G. J. DAVIES et al. ACTA CRYSTALLOGR., SECT.D, Vol. 52, p. 7 1996; G.J. DAVIES et al. BIOCHEMISTRY, V. 34, p. 16210, 1995); SwissProtaccession number P43316, and the sequences shown in SEQ ID 4. Thecorresponding full-length sequence is disclosed in WO 91/17243 and shownhere in SEQ ID NO: 5. The numeration of all description and claims ofthis invention pertain to the core fragment, however, it is contemplatedthat all claims are also valid for the corresponding positions in thefull-length protein.

TABLE 1 Alignment and numeration scheme for subtilisins (SEQ ID NOS: 46,45, 47, 48, 49, 50, 51, 52, 54, 55, 56, 53, respectively) Pro- Pro- Pro-Pro- Pro- BPN′ Alcalase teaseB Esperase teaseC teaseD teaseE teaseAProperase Relase Savinase PD498  −6 W  −5 S  −4 P  −3 N  −2 D  −1 P   1A A A A A A A A A A Y   2 Q Q Q Q Q Q Q Q Q Q Q Y   3 S T T T S S S S SS S S   3a A   4 V V I V V V V V V V V Y   5 P P P P P P P P P P P Q   6Y Y W W W W W W W W W Y   7 G G G G G G G G G G G G   8 V I I I I I I II I I P   9 S P S S S S S S S S S Q  10 Q L R F R R R R R R R N  11 I IV I V V V V V V V T  12 K K Q N Q Q Q Q Q Q Q S  13 A A A T A A A A A AA T  14 P D P Q P P P P P P P P  15 A K A Q A A A A A A A A  16 L V A AA A A A A A A A  17 H Q H H H H H H H H H W  18 S A N N N N N N N N N D 19 Q Q R R R R R R R R R V  20 G G G G G G G G G G G  21 Y F L I L L LL L L L T  22 T K T F T T T T T T T R  23 G G G G G G G G G G G G  24 SA S N S S S S S S S S  25 N N G G G G G G G G G S  26 V V V A V V V V VV V T  27 K K K R R K K K K K K Q  28 V V V V V V V V V V V T  28a V  29A A A A A A A A A A A A  30 V V V V V V V V V V V V  31 I L L L L L L LL L L L  32 D D D D D D D D D D D D  33 S T T T T T T T T T T S  34 G GG G G G G G G G G G  35 I I I I I I I I I I I V  36 D Q D D  37 S A S AS S S S S S S Y  38 S S T S T T T T T T T N  39 H H H H H H H H H H H H 40 P P P P P P P P P P P P  41 D D D D D D D D D D D D  42 L L L L L LL L L L L L  43 K N N R N N N N N N N A  44 V V I I I I I I I I I R  44aK  44b V  45 A V R A R R R R R R R I  46 G G G G G G G G G G G K  47 G GG G G G G G G G G G  48 A A A A A A A A A A A Y  49 S S S S S S S S S SS D  50 M F F F F F F F F F F F  51 V V V I V V V V V V V I  52 P A P SP P P P P P P D  53 S G G S G G G G G G G R  54 E E E E E E E E E E E D 55 T A P P P P P P P P N  56 P Y P N  57 N N S S S S S S S S S P  58 FT Y T T T T T T T M  59 Q T Q H Q Q Q Q Q Q Q  60 D D D D D D D D D D DD  61 D G G N G G G G G G G L  62 N N N N N N N N N N N N  63 S G G G GG G G G G G G  64 H H H H H H H H H H H H  65 G G G G G G G G G G G G 66 T T T T T T T T T T T T  67 H H H H H H H H H H H H  68 V V V V V VV V V V V V  69 A A A A A A A A A A A A  70 G G G G G G G G G G G G  71T T T T T T T T T T T T  72 V V I I I I I I I I I V  73 A A A A A A A AA A A A  74 A A A A A A A A A A A A  75 L L L L L L L L L L L D  75a T 76 N D N N N D N N N D N N  77 N N N N N N N N N N N N  78 S T S S S SS S S S S G  79 I T I I I I I I I I I I  80 G G G G G G G G G G G G  81V V V V V V V V V V V V  82 L L L L L L L L L L L A  83 G G G G G G G GG G G G  84 V V V V V V V V V V V M  85 A A A A A A A A A A A A  86 P PP P P P P P P P P P  87 S S S S S S S S N S S D  88 S V A A A A A A A AA T  89 A S E D E E E E E E E K  90 L L L L L L L L L L L I  91 Y Y Y YY Y Y Y Y Y Y L  92 A A A A A A A A A A A A  93 V V V V V V V V V V V V 94 K K K K K K K K K K K R  95 V V V V V V V V V V V V  96 L L L L L LL L L L L L  97 G N G D G G G G G G G D  98 D S A R A A A A A A A A  99A S S N S S S S S S S N 100 G G G G G G G G G G G G 101 S S S S S S G SG S S S 102 G G G G G G G G G G G G 103 Q S S S S A A S S S S S 104 Y YV L Y I I V N V V L 105 S S S A S S S S S S S D 106 W G S S S S S S S SS S 107 I I I V I I I I I I I I 108 I V A A A A A A A A A A 109 N S Q QQ Q Q Q Q Q Q S 110 G G G G G G G G G G G G 111 I I L I L L L L L L L I112 E E E E E E E E E E E R 113 W W W W W W W W W W W Y 114 A A A A A AA A A A A A 115 I T G I G G G G G G G A 116 A T N N N N N N N N N D 117N N N N N N N N N N N Q 118 N G G N G G G G G G G G 119 M M M M M M M MM M M A 120 D D H H H H H H H D H K 121 V V V I V V V V V V V V 122 I IA I A A A A A A A L 123 N N N N S N N N N N N N 124 M M L M L L L L L LL L 125 S S S S S S S S S S S S 126 L L L L L L L L L L L L 127 G G G GG G G G G G G G 128 G G S S S S S S S S S C 129 P A P T P P P P P P P E130 S S S S S S S S S S S C 131 G G P G P P P A P P P N 132 S S S S S SS G S S S S 133 A T A S A A A G A A A T 134 A A T T T T T T T T T T 135L M L L L L L L L L L L 136 K K E E E E E E E E E K 137 A Q Q L Q Q Q QQ Q Q S 138 A A A A A A A A A A A A 139 V V V V V V V V V V V V 140 D DN N N N N N N N N D 141 K N S R S S S S S S S Y 142 A A A A A A A A A AA A 143 V Y T N T T T T T T T W 144 A A S N S S S S S S S N 145 S R R AR R R R R R R K 146 G G G G G G G G G G G G 147 V V V I V V V V V V V A148 V V L L L L L L L L L V 149 V V V L V V V V V V V V 150 V V V V V VV V V V V V 151 A A A G A A A A A A A A 152 A A A A A A A A A A A A 153A A S A S S S S S S S A 154 G G G G G G G G G G G G 155 N N N N N N N NN N N N 156 E S S T S S S S S S S D 157 G G G G G G G G G G G N 158 S SA R A A A A A A A V 159 T S Q 160 G G G G G G D G G G G S 161 S N S S SS S S S S R 162 S T I I I I I I I I T 163 S N S S S S S S S S F 164 T T165 V I V 166 G G N 167 Y Y Y Y Y Y Y A Y Y Y Q 168 P P P P P P P P P PP P 169 G A A A A A A A A A A A 170 K K R R R R R S R R R S 171 Y Y Y YY Y Y Y Y Y Y Y 172 P D A S A A A A A A A P 173 S S N G N N N N N N N N174 V V A V A A A A A A A A 175 I I M M M M M M M M M I 176 A A A A A AA A A A A A 177 V V V V V V V V V V V V 178 G G G A G G G G G G G G 179A A A A A A A A A A A A 180 V V T V T T T T T T T I 181 D D D D D D D DD D D D 182 S S Q Q Q Q Q Q Q Q Q S 183 S N N N N N N N N N N N 184 N SN G N N N N N N N D 185 Q N N Q N N N N N N N R 186 R R R R R R R R R RR K 187 A A A A A A A A A A A A 188 S S S S S S S S S S S S 189 F F F FF F F F F F F F 190 S S S S S S S S S S S S 191 S S Q T Q Q Q Q Q Q Q N192 V V Y Y Y Y Y Y Y Y Y Y 193 G G G G G G G G G G G G 194 P A A P A AA P A A A T 195 E E G E G G G G G E G W 196 L L L I L L L L L L L V 197D E D E D D D D D D D D 198 V V I I I I I I I I I V 199 M M M S V V V VV V V T 200 A A A A A A A A A A A A 201 P P P P P P P P P P P P 202 G GG G G G G G G G G G 203 V A V V V V V V V V V V 204 S G N N N N N N N NN N 205 I V I V V V V V V V V I 206 Q Y Q N Q Q Q Q Q Q Q A 207 S S S SS S S S S S S S 208 T T T T T T T T T T T T 209 L Y Y Y Y Y Y Y Y Y Y V210 P P P T P P P P P P P P 211 G T G G G G G G G G G N 212 N N S N S SS S S S S N 213 K T T R T T T T T T T G 214 Y Y Y Y Y Y Y Y Y Y Y Y 215G A A V A A A A A A A S 216 A T S S S S S S S S S Y 217 Y L D L L L L LL L L M 218 N N N S N N N N N N N S 219 G G G G G G G G G G G G 220 T TT T T T T T T T T T 221 S S S S S S S S S S S S 222 M M M M M M M M M MM M 223 A A A A A A A A A A A A 224 S S T T T T T T T T T S 225 P P P PP P P P P P P P 226 H H H H H H H H H H H H 227 V V V V V V V V V V V V228 A A A A A A A A A A A A 229 G G G G G G G G G G G G 230 A A A V A AA A A A A L 231 A A A A A A A A A A A A 232 A A A A A A V A A A A A 233L L L L L L L L L L L L 234 I I V V V V V V V V V L 235 L L K K K K K KK L K A 236 S S Q S Q Q H Q Q Q Q S 237 K K K R K K K K K K K Q 238 H HN Y N N N N N N N G 239 P P P P P P P P P P P K 240 N N S S S S S S S SS N 241 W L W Y W W W W W W W 242 T S S T S S S S S S S 243 N A N N N NN N N N N N 244 T S V N V V V V V V V V 245 Q Q Q Q Q Q R Q Q Q Q Q 246V V I I I I I I I I I I 247 R R R R R R R R R R R R 248 S N N Q N N D NN N N Q 249 S R H R H H H H H H H A 250 L L L I L L L L L L L I 251 Q SK N K K K K K K K E 252 N S N Q N N K N N N N Q 253 T T T T T T T T T TT T 254 T A A A A A A A A A A A 255 T T T T T T T T T T T D 256 K Y S YS S S S S S S K 257 L L L L L L L L L L L I 258 G G G G G G G G G G G S259 D S S S S S S S S S S G 260 S S T P T T T T T T T T 261 F F N S N NN N N N N G 262 Y Y L L L L L L L L L T 263 Y Y Y Y Y Y Y Y Y Y Y N 264G G G G G G G G G G G F 264a K 265 K K S N S S S S S S S Y 266 G G G G GG G G G G G G 267 L L L L L L L L L L L K 268 I I V V V V V V V V V I269 N N N H N N N N N N N N 270 V V A A A A A A A A A S 271 Q E E G E EE E E E E N 272 A A A R A A A A A A A K 273 A A A A A A A A A A A A 274A A T T A T T T T T T V 275 Q Q R Q R R R R R R R R 276 Y

EXAMPLES Example 1 Identification of Epitope Sequences and EpitopePatterns

High diversity libraries (10¹²) of phages expressing random hexa-, nona-or dodecapetides as part of their membrane proteins, were screened fortheir capacity to bind purified specific rabbit IgG, and purified ratand mouse IgG1 and IgE antibodies. The phage libraries were obtainedaccording to prior art (se WO 9215679 hereby incorporated by reference).

The antibodies were raised in the respective animals by subcutaneous,intradermal, or intratracheal injection of relevant proteins (e.g.proteases, lipolytic enzymes, amylases, oxidoreductases) dissolved inphosphate buffered saline (PBS). The respective antibodies were purifiedfrom the serum of immunised animals by affinity chromatography usingparamagnetic immunobeads (Dynal AS) loaded with pig anti-rabbit IgG,mouse anti-rat IgG1 or IgE, or rat anti-mouse IgG1 or IgE antibodies.

The respective phage libraries were incubated with the IgG, IgG1 and IgEantibody coated beads. Phages, which express oligopeptides with affinityfor rabbit IgG, or rat or mouse IgG1 or IgE antibodies, were collectedby exposing these paramagnetic beads to a magnetic field. The collectedphages were eluted from the immobilised antibodies by mild acidtreatment, or by elution with intact enzyme. The isolated phages wereamplified as know to the specialist. Alternatively, immobilised phageswere directly incubated with E. coli for infection. In short, F-factorpositive E. coli (e.g. XL-1 Blue, JM101, TG1) were infected withM13-derived vector in the presence of a helper-phage (e.g. M13K07), andincubated, typically in 2xYT containing glucose or IPTG, and appropriateantibiotics for selection. Finally, cells were removed bycentrifugation. This cycle of events was repeated 2-5 times on therespective cell supernatants. After selection round 2, 3, 4, and 5, afraction of the infected E. coli was incubated on selective 2xYT agarplates, and the specificity of the emerging phages was assessedimmunologically. Thus, phages were transferred to a nitrocellulase (NC)membrane. For each plate, 2 NC-replicas were made. One replica wasincubated with the selection antibodies, the other replica was incubatedwith the selection antibodies and the immunogen used to obtain theantibodies as competitor. Those plaques that were absent in the presenceof immunogen, were considered specific, and were amplified according tothe procedure described above.

The specific phage-clones were isolated from the cell supernatant bycentrifugation in the presence of polyethylenglycol. DNA was isolated,the DNA sequence coding for the oligopeptide was amplified by PCR, andthe DNA sequence was determined, all according to standard procedures.The amino acid sequence of the corresponding oligopeptide was deducedfrom the DNA sequence.

Thus, a number of peptide sequences with specificity for the proteinspecific antibodies, described above, were obtained. These sequenceswere collected in a database, and analysed by sequence alignment toidentify epitope patterns. For this sequence alignment, conservativesubstitutions (e.g. aspartate for glutamate, lysine for arginine, serinefor threonine) were considered as one. This showed that most sequenceswere specific for the protein the antibodies were raised against.However, several cross-reacting sequences were obtained from phages thatwent through 2 selection rounds only. In the first round 22 epitopepatterns were identified.

In further rounds of phage display, more antibody binding sequences wereobtained leading to more epitope patterns. Further, the literature wassearched for peptide sequences that have been found to bindenvironmental allergen-specific antibodies (J All Clin Immunol 93 (1994)pp. 34-43; Int Arch Appl Immunol 103 (1994) pp. 357-364; Clin ExpAllergy 24 (1994) pp. 250-256; Mol Immunol 29 (1992) pp. 1383-1389; JImmunol 121 (1989) pp. 275-280; J. Immunol 147 (1991) pp. 205-211; MolImmunol 29 (1992) pp. 739-749; Mol Immunol 30 (1993) pp. 1511-1518; MolImmunol 28 (1991) pp. 1225-1232; J. Immunol 151 (1993) pp. 7206-7213).These antibody binding peptide sequences were included in the database.

A first generation database of antibody binding peptides identified andtheir corresponding epitope patterns are shown in Table 2-7 below.

Tables 2-7: Overview of the antibody binding peptide sequences, epitopepatterns and epitope sequences. The type of antibody used foridentifying the antibody binding sequences is indicated as IgG or IgEand the species from which the antibodies were derived are indicated asmo (mouse), ra (rat) and hu (human).

TABLE 2 Savinase antibody binding peptide sequences, epitope patternsand epitope sequences. Antibody binding Method of Epitope peptideidentification pattern Donor Acceptor Epitope Sequence (BPN') VQVYGDTSA(SEQ Phage Q > Y > D > Savinase savinase Q206 V81 Y214 G80 D41 ID NO:59) display T208 LQCVGS (SEQ ID Protein a-amylase savinase L21 Q236 V26G25 S24 NO: 60) fragments inhibitor KRFANTELA (SEQ Phage R/K R F > NSavinase savinase K251 R247 A174 N173 ID NO: 61) display LDQIFFTRW (SEQPhage D/E Q I F F T Savinase savinase L42/L75 D41 Q2 I79 ID NO: 62)display FNDAFFVKM (SEQ Phage Savinase savinase N185 D181 A187 F189 IDNO: 63) display V203 ANIPIWSRSA (SEQ Phage > R S A Savinase savinaseR145 S144 A142 ID NO: 64) display ANIPIWSRSA (SEQ Phage > R S A Savinasesavinase S188 R186 S190 A179 ID NO: 64) display RQSTDFGTT (SEQ Phage RQ > > Savinase savinase R186 Q191 S156 ID NO: 65) display D/E VQVYGDTSA(SEQ Phage Q > Y > D > Savinase savinase Q191 Y192 ID NO: 66) displayG193/A194/G195 D197 S265 RRFSNATRA (SEQ Phage R/K R F > N Savinasesavinase K251 R247 A174 N173 ID NO: 67) display CTARLRAGNACG Phage AR > A Savinase savinase A172/A169 R170 A194 (SEQ ID NO: 68) display G193N261 LDQIFFTRW (SEQ Phage D/E Q I F F T Savinase savinase D60 Q59I44/I35 ID NO: 69) display LDQIFFTRW (SEQ Phage D/E Q I F F T Savinasesavinase L42/L75 D41 Q2 I79 ID NO: 69) display EQIFFTSGL (SEQ ID PhageD/E Q I F F T Savinase savinase E112 Q109 I79 NO: 70) display GRFSNSKFK(SEQ Phage L > G R S Savinase savinase L196 G195 R170 S163 ID NO: 71)display AVLRDC (SEQ ID Protein a-amylase savinase A254 V268 L267 R10D181 NO: 72) fragments inhibitor LQCVGS (SEQ ID Protein a-amylasesavinase L217 Q206 V81 G80 S3 NO: 73) fragments inhibitor LRQCNERCV (SEQPhage R Q > > Savinase savinase L267 R10 Q12 N269 E271 ID NO: 74)display D/E R275 SPVTKRASLKIDSKK Protein Der p II savinase A88 S87/T22L233 K235 (SEQ ID NO: 75) fragments I246 RQSTDFGTT (SEQ Phage R Q > >Savinase savinase R247 Q245 S240/S242 ID NO: 76) display D/E FCTNNCELS(SEQ Phage N > > E L Savinase savinase T143 N173 N140 E136 L135 ID NO:77) display FCTNNCELS (SEQ Phage N > > E L Savinase savinase N117 N116E112 L111 ID NO: 77) display DFHVKYAAQ (SEQ Phage Savinase savinase IDNO: 78) display VAQYKALPVVLENA Protein Fel d I savinase L135 P168 V139L111 E112 (SEQ ID NO: 79) fragments N116 AAYPDV (SEQ ID ProteinA > > > > Y a-amylase savinase A215 Y214 P40 D41 V81 NO: 80) fragmentsP > inhibitor EQIFFTSGL (SEQ ID Phage D/E Q I F F T Savinase savinaseE271 Q12 I8 NO: 81) display VDAAF (SEQ ID NO: Protein Poa p IX savinaseV203 D181 A179 A187 F189 82) fragments AVLRDC (SEQ ID Protein a-amylasesavinase A232 V234 L250 R247 D197 NO: 83) fragments inhibitor RAFRRNANW(SEQ Phage A R > A Savinase savinase A272/A273 R275 R19 N18 ID NO: 84)display A15/A16 CTARLRAGNACG Phage A R > A Savinase savinase A15/A16 R19L21 R275 (SEQ ID NO: 85) display A272 A273 N269 TFHDAPALQ (SEQ PhageSavinase savinase H39 D41 A74/A73 P86 A88 ID NO: 86) display L90CTARVVALGVCG Phage A R > A Savinase savinase R145 V147 V149 A151 (SEQ IDNO: 87) display L124/L126 G127 GRFSNSKFK (SEQ Phage L > G R S Savinasesavinase L148 G146 R145 ID NO: 88) display S144/S141 N140 RRFANDHTR (SEQPhage R/K R F > N savinase savinase K27 R45 N43 D41 H39 ID NO: 89)display T38/T213 KRFANTEPA (SEQ Phage R/K R F > N savinase savinase K251R247 A174 N173 ID NO: 90) display YKVSAL (SEQ ID Protein a-amylasesavinase Y91 K27 V26 S24 G23 L21 NO: 91) fragments inhibitor TGKYVS (SEQID Protein a-amylase savinase S24 G25 K27 Y91 V93 NO: 92) fragmentsinhibitor Antibody binding peptide Epitope # IgG IgE VQVYGDTSA (SEQsav1.1 Ra ID NO: 59) LQCVGS (SEQ ID sav19.1 Hu NO: 60) KRFANTELA (SEQsav6.1 Ra- Mo ID NO: 61) Mo LDQIFFTRW (SEQ sav5.1 Ra ID NO: 62)FNDAFFVKM (SEQ sav11.0 Ra ID NO: 63) ANIPIWSRSA (SEQ sav3.2-lac1.0- RaID NO: 64) lip4.0-pd5.0 ANIPIWSRSA (SEQ sav3.1-lac1.0- Ra ID NO: 64)lip4.0-pd5.0 RQSTDFGTT (SEQ sav2.2 Ra ID NO: 65) VQVYGDTSA (SEQ sav1.2Ra ID NO: 66) RRFSNATRA (SEQ sav6.1 Ra- Mo ID NO: 67) Mo CTARLRAGNACGsav10.4 Ra (SEQ ID NO: 68) LDQIFFTRW (SEQ sav5.2 Ra ID NO: 69) LDQIFFTRW(SEQ sav5.1 Ra ID NO: 69) EQIFFTSGL (SEQ ID sav5.4 Ra NO: 70) GRFSNSKFK(SEQ sav9.2-je4.0-lip5.1-5.2 Ra ID NO: 71) AVLRDC (SEQ IDsav18.1-pd18.1-18.2 Hu NO: 72) LQCVGS (SEQ ID sav19.2 Hu NO: 73)LRQCNERCV (SEQ sav2.1 Ra ID NO: 74) SPVTKRASLKIDSKK sav16.0-pd7.0 Hu(SEQ ID NO: 75) RQSTDFGTT (SEQ sav2.3 Ra ID NO: 76) FCTNNCELS (SEQsav7.2 Ra ID NO: 77) FCTNNCELS (SEQ sav7.1 Ra ID NO: 77) DFHVKYAAQ (SEQsav8.0 Ra ID NO: 78) VAQYKALPVVLENA sav12.0-pd8.0 Hu (SEQ ID NO: 79)AAYPDV (SEQ ID sav13.0-pd13.1-13.2 Hu NO: 80) EQIFFTSGL (SEQ ID sav5.3Ra NO: 81) VDAAF (SEQ ID NO: sav15.0-pd12.0 Hu 82) AVLRDC (SEQ IDsav18.2-pd18.1-18.2 Hu NO: 83) RAFRRNANW (SEQ sav10.1 Ra ID NO: 84)CTARLRAGNACG sav10.2 Ra (SEQ ID NO: 85) TFHDAPALQ (SEQ sav4.0 Ra ID NO:86) CTARVVALGVCG sav10.3 Ra (SEQ ID NO: 87) GRFSNSKFK (SEQsav9.1-je4.0-lip5.1-5.2 Ra ID NO: 88) RRFANDHTR (SEQ sav6.2 Ra ID NO:89) KRFANTEPA (SEQ sav6.1 Ra- Mo ID NO: 90) Mo YKVSAL (SEQ IDsav14.0-pd14.0 Hu NO: 91) TGKYVS (SEQ ID sav17.0-pd17.1-17.2 Hu NO: 92)

TABLE 3 PD498 antibody binding peptide sequences, epitope patterns andepitope sequences. Epitope pattern Donor Acceptor Epitope Sequence(BPN′) Epitope # IgG IgE Fel d I pd498 V198 A254 Q252 Y276 K239 A235L233 P86 pd8.0 Hu A > > > a-amylase pd498 *3aA Y1/Y2 P-4/P-1 D-2 V81pd13.2 Hu > Y P > inhibitor Poa p IX pd498 S182 Y6 G7 P8 T13 P14 A15 A16pd11.0 Hu > K L > > Poa p IX pd498 Y171 K136 L135 A108 Y113 pd4.4 Hua-amylase pd498 Y48/Y37 K46 *44aaV A43 L42 pd14.0 Hu inhibitor Poa p IXpd498 V196/V198 D197 A174/A176 A169 F163 pd12.0 Hu K Q S Poa p IX pd498A142 A147 V148 K120 Q27 S24/S25 pd2.3 Hu K Q S pd498 pd498 R44 K89 Q27S236 K120 G146 pd2.2 Ra Der p II pd498 *28aV T88 *44a K R44 A43 L42pd7.0 Hu > K L > > pd498 pd498 N56/N55 K46 L91 A29/A119 T28 pd4.3 Ra > KL > > pd498 pd498 N240/N243 K239 L233/L234 A16 T21 R22 pd4.1 Ra > KL > > Poa p IX pd498 Y37 K46 L91 A114 Y113 pd4.5 Hu > K L > > pd498pd498 N240/N243 K239 L233/L234 A16 T21 R22 pd4.1 Ra Y I > K L pd498pd498 Y113 I111 A108/A138 K136 L135 pd3.1 Ra K Q S pd498 pd498 A115 K145N243 N240 K239 Q237 S236 pd2.1 Ra > R Y > K/R pd498 pd498 R94 R53 Y48Q117 R112 S109/S137 pd1.5-lac2.0 Ra Phl p V pd498 A169 Q167 F163 T162S160 G193 pd10.0 Hu Y I > K L pd498 pd498 Y276 I246 K239 L234 S236 pd3.2Ra > K L > > pd498 pd498 N240/N243 K239 L233/L234 R22 P86 pd4.2 RaA > > > a-amylase pd498 *3aA Y2 P14 D18 V19 pd13.1 Hu > Y P > inhibitorK Q S Poa p IX pd498 A15 A16 V274 K239 Q237 S236 pd2.4 Hu a-amylasepd498 G146 K145 Y141 V139 S137 pd17.2 Hu inhibitor a-amylase pd498 A273V274 L233 R22 D87 pd18.1 Hu inhibitor A R > A Par j 1 + pd498 N10 S12A15/A16 R275 A273/A249 R247 pd9.0 Hu + Ra Hu Par o 1 A174 D197 S170pd498 pd498 R22 G23 L233 S236 pd6.2 Ra > R Y > K/R pd498 pd498 R94 R53Y48 P57 K46 L91 pd1.4-lac2.0 Ra > R Y > K/R pd498 pd498 R94 R53 Y48 P57K46 L91 pd1.4-lac2.0 Ra a-amylase pd498 L96 R94 S33 V35 Y37 pd15.0 Huinhibitor > R Y > K/R pd498 pd498 S109/S137 R112 Y141 N144 K145pd1.3-lac2.0 Ra > R Y > K/R pd498 pd498 T162 R161 Y192 N191 K186pd1.2-lac2.0 Ra > R Y > K/R pd498 pd498 T133/T134 R112 Y141 N144 K145pd1.1-lac2.0 Ra a-amylase pd498 A92 *44aaV L42 R44 D75 pd18.2 Huinhibitor a-amylase pd498 S236 G238 K239 Y276 V274 S270 pd17.1 Huinhibitor a-amylase pd498 S12 P14 W17 S-5 W-6 pd16.0 Hu inhibitor > R SA pd498 pd498 S137 R112 S109 A108 pd5.0-lac1.0-lip4.0- Ra sav3.1-3.2pd498 pd498 S215 M217 I205 M222 G219 pd6.1 Ra

TABLE 4 Antibody binding peptide sequences, epitope patterns and epitopesequences for the T. lanuginosus lipase (Lipolase). Antibody bindingMethod of Epitope peptide identification pattern Donor Acceptor EpitopeSequence Epitope # IgG IgE QRPPRYELE (SEQ Phage R P P R lipolaselipolase lip1.0 Ra ID NO: 93) display ELEYRPPRQ (SEQ Phage > E Ylipolase lipolase L124 E129 Y164 lip2.1 Ra ID NO: 94) display HEYDMRVAW(SEQ Phage > E Y lipolase lipolase H215 E219 Y220 lip2.2 Ra ID NO: 95)display HEYPMDFHL (SEQ Phage > E Y lipolase lipolase H215 E219 Y220lip2.2 Ra ID NO: 96) display SEYSMSITP (SEQ Phage > E Y lipolaselipolase S217 E219 Y220 lip2.3 Ra ID NO: 97) display CVWPAHAPLSCGPhage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (SEQ ID NO:98) display A P > S P208/P207 S214/S216/S217 CSWPSPAPLSCGPhage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (SEQ ID NO:99) display A P > S P208/P207 S214/S216/S217 CDFPLHAPLSCGPhage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (SEQ ID NO:100) display A P > S P208/P207 S214/S216/S217 CLFPSPAPRSCGPhage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (SEQ ID NO:101) display A P > S P208/P207 S214/S216/S217 CDGPAPAPWSCGPhage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (SEQ ID NO:102) display A P > S P208/P207 S214/S216/S217 CSFPLPAPRSCGPhage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (SEQ ID NO:103) display A P > S P208/P207 S214/S216/S217 CVYPSPAPWSCGPhage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (EQ ID NO:104) display A P > S P208/P207 S214/S216/S217 PEYTMNALS (SEQ Phage > E Ylipolase lipolase P218 E219 Y220 lip2.4 Ra ID NO: 105) displayCSRSAKGARLCG Phage > R S A lipolase lipolase R209 S214 A182lip4.0-lac1.0- Ra (EQ ID NO: 106) display pd5.0- sav3.1-3.2 LEYPMSASQ(SEQ Phage > E Y lipolase lipolase L124 E129 Y164 lip2.1 Ra ID NO: 107)display RKLTLSGRS (SEQ Phage L > G R S lipolase lipolase L67 G65 R81S83/S85 lip5.1-je4.0- Ra ID NO: 108) display sav9.0 RKLTLSGRS (SEQ PhageL > G R S lipolase lipolase L96/L97 G212 R209/ lip5.2-je4.0- Ra ID NO:109) display R179 S214 sav9.0 SYGAPATPAA (SEQ Protein Poa p IX lipolaseS170 Y171 G172 A173 lip6.0 Hu ID NO: 110) fragments P174 A150 T153PAAGYTPAAP (SEQ Protein Poa p IX lipolase A18/A19/A20 G65 Y53 lip7.0 HuHu ID NO: 111) fragments T123 YKLAY (SEQ ID NO: Protein Poa p IXlipolase Y138 K74 L75 A68 Y16 lip8.1 Hu 112) fragments YKLAY (SEQ ID NO:Protein Poa p IX lipolase Y53 K127 L67 A68 Y16 lip8.2 Hu 112) fragmentsKYDDYVATLS (SEQ Protein Poa p IX lipolase Y194 D167 D165 Y164 lip9.0 HuID NO: 113) fragments V132 A131 L52 S54 EVKATPAGEL (SEQ Protein Poa p IXlipolase E43 V44 K46 A47 T72 lip10.0 Hu ID NO: 114) fragmentsCGYSNAQGVDYWI Protein Der p I lipolase Y53 S54 N25/N26 lip15.0 Hu Hu(SEQ ID NO: 115) fragments A18/A19/A20 Q15 V44 VPGIDPNACHYMKC ProteinDer p II lipolase P256 I255 D254 P253 lip16.0 Hu (SEQ ID NO: 116)fragments N200 H198 Y261 SPVTKRASLKIDSKK Protein Der p II lipolase R179A182 S216/S217 lip17.0 Hu (SEQ ID NO: 117) fragments I238 K237 I235 D234S224 K223 IMSALAMVYLGAK Protein Ovalbumin lipolase V140 Y138 L69 A49 A47lip18.0 Hu (SEQ ID NO: 118) fragments K46 ELGVRE (SEQ ID Proteina-amylase lipolase E99 L97 G109/G177 lip11.0 Hu NO: 119) fragmentsinhibitor V176 R175 D242 GCRKEV (SEQ ID Protein a-amylase lipolase G106C107 R108 K98 lip12.0 Hu NO: 120) fragments inhibitor E99 LRSVYQ (SEQ IDProtein a-amylase lipolase L147 R81 S79 V77 Y16 lip13.0 Hu NO: 121)fragments inhibitor Q15 SGPWSW (SEQ ID Protein a-amylase lipolase S170G172 P174 W89 lip14.0 Hu NO: 122) fragments inhibitor S83

TABLE 5 Amylase (Natalase) antibody binding peptide sequences, epitopepatterns and epitope sequences. Antibody binding Method of peptideidentification Epitope pattern Donor Acceptor Epitope Sequence ARIDPRGPS(SEQ Phage A > I D P R/K amylase amylase A380 K381 I382 D383 P384 ID NO:123) display R389 ARIDPRHGS (SEQ Phage A > I D P R/K amylase amylaseA380 K381 1382 D383 P384 ID NO: 124) display R389 CSVAKIDPRTCG PhageA > I D P R/K amylase amylase A109 K138 D140 P142 (SEQ ID NO: 125)display R144 CSVAKIDPRTCG Phage A > 1 D P R/K amylase amylase A380 K381I382 D383 P384 (SEQ ID NO: 125) display R389 AKIDPKPDT (SEQ Phage A > ID P R/K amylase amylase A109 K138 D140 P142 ID NO: 126) display R144AKIDPKPDT (SEQ Phage A > I D P R/K amylase amylase A380 K381 I382 D383P384 ID NO: 126) display R389 ARIDPRHGS (SEQ Phage A > I D P R/K amylaseamylase A109 K138 D140 P142 ID NO: 127) display R144 QIYNDTGPT (SEQPhage Q > Y > D > amylase amylase Q390 L386 Y368/Y367 D366 ID NO: 128)display QIYNDTGPT (SEQ Phage Q > Y > D > amylase amylase Q170 I173 Y196D195 ID NO: 128) display QIYNDTGPT (SEQ Phage Q > Y > D > amylaseamylase Q357 I352 Y349 D366 ID NO: 128) display QIYNDTGPT (SEQ PhageQ > Y > D > amylase amylase Q331 I370 Y368/Y367 D366 ID NO: 128) displayCGSATIDPRQCG Phage A > I D P R/K amylase amylase A109 K138 D140 P142(SEQ ID NO: 129) display R144 CNADNQMYPQCG Phage A > > > > Y P > amylaseamylase N29 A27 D26/D25 Y8 (SEQ ID NO: 130) display P41/P42 ARIDPRGPS(SEQ Phage A > I D P R/K amylase amylase A109 K138 D140 P142 ID NO: 131)display R144 CGSATIDPRQCG Phage A > I D P R/K amylase amylase A380 K381I382 D383 P384 (SEQ ID NO: 132) display R389 CDADSSGYPLCG PhageA > > > > Y P > amylase amylase A107/A109 D108 Y57 (SEQ ID NO: 133)display P41/42 QLYGDEQLP (SEQ Phage Q > Y > D > amylase amylase Q331I370 Y368/Y367 D366 ID NO: 134) display QLYGDEQLP (SEQ Phage Q > Y > D >amylase amylase Q357 I352 Y349 D366 ID NO: 134) display QLYGDEQLP (SEQPhage Q > Y > D > amylase amylase Q170 I173 Y196 D195 ID NO: 134)display QLYGDEQLP (SEQ Phage Q > Y > D > amylase amylase Q390 L386Y368/Y367 D366 ID NO: 134) display RYAQIDPRW (SEQ Phage A > I D P R/Kamylase amylase A380 K381 I382 D383 P384 ID NO: 135) display R389RYAQIDPRW (SEQ Phage A > I D P R/K amylase amylase A109 K138 D140 P142ID NO: 135) display R144 GEFNLGRSS (SEQ Phage L > G R S amylase amylaseL88 G92 R31 S28 ID NO: 136) display CNADSWGYPRCG Phage A > > > > Y P >amylase amylase N29 A27 D26/D25 Y8 (SEQ ID NO: 137) display P41/P42CNADNQMYPQCG Phage A > > > > Y P > amylase amylase N102 A233 D232 Y54(SEQ ID NO: 138) display P41/P42 CNADSWGYPRCG Phage A > > > > Y P >amylase amylase N102 A233 D232 Y54 (SEQ ID NO: 137) display P41/P42GEFNLGRSS (SEQ Phage L > G R S amylase amylase L62 G63/G76 R78 S79 IDNO: 139) display Antibody binding peptide Epitope # IgG IgE ARIDPRGPS(SEQ je1.1 Ra ID NO: 123) ARIDPRHGS (SEQ je1.1 Ra ID NO: 124)CSVAKIDPRTCG je1.2 Ra (SEQ ID NO: 125) CSVAKIDPRTCG je1.1 Ra (SEQ ID NO:125) AKIDPKPDT (SEQ je1.2 Ra ID NO: 126) AKIDPKPDT (SEQ je1.1 Ra ID NO:126) ARIDPRHGS (SEQ je1.2 Ra ID NO: 127) QIYNDTGPT (SEQ je2.4 Ra ID NO:128) QIYNDTGPT (SEQ je2.3 Ra ID NO: 128) QIYNDTGPT (SEQ je2.2 Ra ID NO:128) QIYNDTGPT (SEQ je2.1 Ra ID NO: 128) CGSATIDPRQCG je1.2 Ra (SEQ IDNO: 129) CNADNQMYPQCG je3.1 Ra (SEQ ID NO: 130) ARIDPRGPS (SEQ je1.2 RaID NO: 131) CGSATIDPRQCG je1.1 Ra (SEQ ID NO: 132) CDADSSGYPLCG e3.3 Ra(SEQ ID NO: 133) QLYGDEQLP (SEQ je2.1 Ra ID NO: 134) QLYGDEQLP (SEQje2.2 Ra ID NO: 134) QLYGDEQLP (SEQ je2.3 Ra ID NO: 134) QLYGDEQLP (SEQje2.4 Ra ID NO: 134) RYAQIDPRW (SEQ je1.1 Ra ID NO: 135) RYAQIDPRW (SEQje1.2 Ra ID NO: 135) GEFNLGRSS (SEQ je4.1-sav9.0-lip5.1-5.2 Ra ID NO:136) CNADSWGYPRCG je3.1 Ra (SEQ ID NO: 137) CNADNQMYPQCG je3.2 Ra (SEQID NO: 138) CNADSWGYPRCG je3.2 Ra (SEQ ID NO: 137) GEFNLGRSS (SEQje4.2-sav9.0-lip5.1-5.2 Ra ID NO: 139)

TABLE 6 Cellulase (Carezyme; Cel45 from Humicola insolens) antibodybinding peptide sequences, epitope patterns and epitope sequences.Antibody binding Method of peptide identification Epitope pattern DonorAcceptor Epitope Sequence Epitope # IgG IgE CVHAGPRAGTCG Phagedisplay > G > > A G carezyme carezyme P23 R201 A83 G84 car1.1 Ra (SEQ IDNO: 140) CVHAGPRAGTCG Phage display V H > G > carezyme carezyme car2.0Ra (SEQ ID NO: 140) CLSGPLAGRVCG Phage display > G > > A G carezymecarezyme P23 R201 A83 G84 car1.1 Ra (SEQ ID NO: 141) CRISPWYSVPCG Phagedisplay carezyme carezyme car3.0 Ra (SEQ ID NO: 142) CLSGPAAGQSCG Phagedisplay > G > > A G carezyme carezyme P23 R201 A83 G84 car1.1 Ra (SEQ IDNO: 143) Phage display A > I D P R/K je-1 carezyme R146 I131 D133car11.2 Ra P137 CITRGTRAGWCG Phage display > G > > A G carezyme carezymeP23 R201 A83 G84 car1.1 Ra (SEQ ID NO: 144) Phage display A R > Asavinase carezyme A191 R200 R201 car6.2 Ra A83 N81 CLSGPAAGQSCG Phagedisplay > G > > A G carezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ IDNO: 143) A100 G101 Phage display A > I D P R/K je-1 carezyme A195 R37I38 D40 car11.1 Ra L44 Phage display Q > Y > D > savinase, carezyme Q59Y54 G134 D133 car10.0 Ra T136 je-1 Phage display > P > > A P > Slipoprime carezyme W62/W169 P61 P165 car9.0 Ra A162 P160 CITRGTRAGWCGPhage display > G > > A G carezyme carezyme T95/S96 G27 P98 car1.2 Ra(SEQ ID NO: 144) A100 G101 Phage display R/K R F > N savinase carezymeR7 R170 F174 car7.0 Ra A177 CLSGPLAGRVCG Phage display > G > > A Gcarezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 145) A100 G101Phage display A R > A savinase carezyme A1 R4 R7 A177 car6.1 Ra N176Phage display > P > R D T G laccase carezyme D178 P180 R4 D2 car5.0 RaS183 Phage display > R Y > K/R pd498 carezyme R170 R153 Y168 P165 car4.0Ra K164 L163 Phage display D/E Q I F F T savinase carezyme Q36 I38 F41F29 car8.0 Ra T197 CLTAGPSAGYCG Phage display > G > > A G carezymecarezyme T95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 146) A100 G101CYTTGRLAGLCG Phage display > G > > A G carezyme carezyme P23 R201 A83G84 car1.1 Ra (SEQ ID NO: 147) CYTTGRLAGLCG Phage display > G > > A Gcarezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 147) A100 G101CVHSGPRAGYCG Phage display > G > > A G carezyme carezyme P23 R201 A83G84 car1.1 Ra (SEQ ID NO: 148) CVHSGPRAGYCG Phage display V H > G > carezyme carezyme car2.0 Ra (SEQ ID NO: 148) CVHAGPRAGTCG Phagedisplay > G > > A G carezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ IDNO: 149) A100 G101 CVHSGPRAGYCG Phage display > G > > A G carezymecarezyme T95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 148) A100 G101CVHSGLSRRLLR Phage display V H > G >  carezyme carezyme car2.0 Ra (SEQID NO: 150) CVTRGPNAGSCG Phage display > G > > A G carezyme carezymeT95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 151) A100 G101 CLTAGPSAGYCG Phagedisplay > G > > A G carezyme carezyme P23 R201 A83 G84 car1.1 Ra (SEQ IDNO: 152) CVTRGPNAGSCG Phage display > G > > A G carezyme carezyme P23R201 A83 G84 car1.1 Ra (SEQ ID NO: 151) CITSGPRAGNCG Phagedisplay > G > > A G carezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ IDNO: 153) A100 G101 CITSGPRAGNCG Phage display > G > > A G carezymecarezyme P23 R201 A83 G84 car1.1 Ra (SEQ ID NO: 153)

TABLE 7 Laccase (Myceliophthora thermopila laccase) antibody bindingpeptide sequences, epitope patterns and epitope sequences. Antibodybinding Method of Epitope peptide identification pattern Donor AcceptorEpitope Sequence Epitope # IgG IgE PQSD5PGESQ (SEQ Phage display P > S/TD P G laccase laccase P180 R175 T168 D166 lac3.2 Ra ID NO: 154) P165G265 WPKSDAGDS (SEQ ID Phage display P > > D A G laccase laccase P241R409 S410/S416 D434 lac4.1 Ra NO: 155) A389 G390 PQSDAGVVM (SEQ ID Phagedisplay P > > D A G laccase laccase P241 R409 S410/S416 D434 lac4.1 RaNO: 156) A389 G390 DPVRDTGAG (SEQ ID Phage display > P > R D T G laccaselaccase P241 R409 D434 T432 lac5.1 Ra NO: 157) G430/G390 GPSRDAGLL (SEQID Phage display P > > D A G laccase laccase P241 R409 S410/S416 D434lac4.1 Ra NO: 158) A389 G390 PASDAGRGP (SEQ ID Phage display P > > D A Glaccase laccase P241 R409 S410/S416 D434 lac4.1 Ra NO: 159) A389 G390PRDSTGLAL (SEQ ID Phage display P > S/T D P G laccase laccase P378 R379T442 D443 lac3.1 Ra NO: 160) P445 G446 PQSDPGESQ (SEQ ID Phage displayP > S/T D P G laccase laccase P378 R379 T442 D443 lac3.1 Ra NO: 161)P445 G446 RYPFLRATN (SEQ ID Phage display > R Y > K/R laccase laccaselac2.0- Ra NO: 162) pd1.1-1.4 GAARDARSA (SEQ ID Phage display > R S Alaccase laccase lac1.0- Ra NO: 163) lip4.0- pd5.0- sav3.1- 3.2 PRSDTGFGS(SEQ ID Phage display > P > R D T G laccase laccase P241 R409 D434 T432lac5.1 Ra NO: 164) G430/G390 LPRSDPGGR (SEQ ID Phage display P > S/T D PG laccase laccase P180 R175 T168 D166 lac3.2 Ra NO: 165) P165 G265DPARDTGDV (SEQ ID Phage display > P > R D T G laccase laccase P241 R409D434 T432 lac5.1 Ra NO: 166) G430/G390 APKSDNGIT (SEQ ID Phage displayP > > D A G laccase laccase P241 R409 S410/S416 lac4.1 Ra NO: 167) D434A389 G390 PKSDPGTNW (SEQ ID Phage display P > S/T D P G laccase laccaseP378 R379 T442 D443 lac3.1 Ra NO: 168) P445 G446 PRTDPGWLA (SEQ ID Phagedisplay P > S/T D P G laccase laccase P378 R379 T442 D443 lac3.1 Ra NO:169) P445 G446 LPRSDPGGR (SEQ ID Phage display P > S/T D P G laccaselaccase P378 R379 T442 D443 lac3.1 Ra NO: 170) P445 G446 PSSDPGARS (SEQID Phage display P > S/T D P G laccase laccase P180 R175 T168 D166lac3.2 Ra NO: 171) P165 G265 HVFDKNVTR (SEQ ID Phage display laccaselaccase lac6.0 NO: 172) PRSDPGTPT (SEQ ID Phage display P > S/T D P Glaccase laccase P378 R379 T442 D443 lac3.1 Ra NO: 173) P445 G446PRSDPGTPT (SEQ ID Phage display P > S/T D P G laccase laccase P180 R175T168 D166 lac3.2 Ra NO: 173) P165 G265 PRDSTGLAL (SEQ ID Phage displayP > S/T D P G laccase laccase P180 R175 T168 D166 lac3.2 Ra NO: 174)P165 G265 PRTDPGWLA (SEQ ID Phage display P > S/T D P G laccase laccaseP180 R175 T168 D166 lac3.2 Ra NO: 175) P165 G265 PSSDPGARS (SEQ ID Phagedisplay P > S/T D P G laccase laccase P378 R379 T442 D443 lac3.1 Ra NO:176) P445 G446 PKSDPGTNW (SEQ ID Phage display P > S/T D P G laccaselaccase P180 R175 T168 D166 lac3.2 Ra NO: 177) P165 G265 WPKSDAGDS (SEQID Phage display P > > D A G laccase laccase P350 S349 D80 A79 G78lac4.2 Ra NO: 178) PQSDAGVVM (SEQ ID Phage display P > > D A G laccaselaccase P350 S349 D80 A79 G78 lac4.2 Ra NO: 179) GPSRDAGLL (SEQ ID Phagedisplay P > > D A G laccase laccase P350 S349 D80 A79 G78 lac4.2 Ra NO:180) PASDAGRGP (SEQ ID Phage display P > > D A G laccase laccase P350S349 D80 A79 G78 lac4.2 Ra NO: 181) APKSDNGIT (SEQ ID Phage displayP > > D A G laccase laccase P350 S349 D80 A79 G78 lac4.2 Ra NO: 182)WPKSDAGDS (SEQ ID Phage display P > > D A G laccase laccase P300 R234S211 D213 A296 lac4.3 Ra NO: 183) PQSDAGVVM (SEQ ID Phage displayP > > D A G laccase laccase P300 R234 S211 D213 A296 lac4.3 Ra NO: 184)GPSRDAGLL (SEQ ID Phage display P > > D A G laccase laccase P300 R234S211 D213 A296 lac4.3 Ra NO: 185) PASDAGRGP (SEQ ID Phage displayP > > D A G laccase laccase P300 R234 S211 D213 A296 lac4.3 Ra NO: 186)APKSDNGIT (SEQ ID Phage display P > > D A G laccase laccase P300 R234S211 D213 A296 lac4.3 Ra NO: 187) DPVRDTGAG (SEQ ID Phagedisplay > P > R D T G laccase laccase P378 R379 D469 T473 G446 lac5.2 RaNO: 188) PRSDTGFGS (SEQ ID Phage display > P > R D T G laccase laccaseP378 R379 D469 T473 G446 lac5.2 Ra NO: 189) DPARDTGDV (SEQ ID Phagedisplay > P > R D T G laccase laccase P378 R379 D469 T473 G446 lac5.2 RaNO: 190) DPVRDTGAG (SEQ ID Phage display > P > R D T G laccase laccaseP60 R59 D51/D53 lac5.3 Ra NO: 191) T10/T12 G30 PRSDTGFGS (SEQ ID Phagedisplay > P > R D T G laccase laccase P60 R59 D51/D53 lac5.3 Ra NO: 192)T10/T12 G30 DPARDTGDV (SEQ ID Phage display > P > R D T G laccaselaccase P60 R59 D51/D53 lac5.3 Ra NO: 193) T10/T12 G30 DPVRDTGAG (SEQ IDPhage display > P > R D T G laccase laccase P157/P155 R23 lac5.4 Ra NO:194) D118 T114 G113 PRSDTGFGS (SEQ ID Phage display > P > R D T Glaccase laccase P157/P155 R23 lac5.4 Ra NO: 195) D118 T114 G113DPARDTGDV (SEQ ID Phage display > P > R D T G laccase laccase P157/P155R23 lac5.4 Ra NO: 196) D118 T114 G113

Example 2 Localisation of Epitope Sequences and Epitope Areas on the3D-Structure of Acceptor Proteins

Epitope sequences were assessed manually on the screen on the3D-structure of the protein of interest, using appropriate software(e.g. SwissProt Pdb Viewer, WebLite Viewer).

In a first step, the identified epitope patterns were fitted with the3D-structure of the enzymes. A sequence of at least 3 amino acids,defining a specific epitope pattern, was localised on the 3D-structureof the acceptor protein. Conservative mutations (e.g. aspartate forglutamate, lysine for arginine, serine for threonine) were considered asone for those patterns for which phage display had evidenced suchexchanges to occur. Among the possible sequences provided by the proteinstructure, only those were retained where the sequence matched a primarysequence, or where it matched a structural sequence of amino acids,where each amino acid was situated within a distance of 5 Å from thenext one. Occasionally, the mobility of the amino acid side chains, asprovided by the software programme, had to be taken in to considerationfor this criterium to be fulfilled.

Secondly, the remaining anchor amino acids as well as the variable aminoacids, i.e. amino acids that were not defining a pattern but werepresent in the individual sequences identified by phage libraryscreening, were assessed in the area around the various amino acidsequences localised in step 1. Only amino acids situated within adistance of 5 Å from the next one were included.

Finally, an accessibility criterium was introduced. The criterium wasthat at least half of the anchor amino acids had a surface that was >30%accessible. Typically, 0-2 epitopes were retained for each epitopepattern. In some cases, two different amino acids could with equalprobability be part of the epitope (e.g. two leucines located close toeach other in the protein 3D-structure). For example, in Savinase twoepitopes actually fit to the antibody binding peptide LDQIFFTRW (SEQ IDNO:62): L75 D41 Q2 179 and L42 D41 Q2 I79. A shorthand notation for sucha situation is: L42/L75 D41 Q2 I79.

Thus, a number of epitope sequences were identified and localised on thesurface of various proteins. As suggested by sequence alignment of theantibody binding peptides, structural analysis confirmed most of theepitopes to be enzyme specific, with only few exceptions. Overall, mostof the identified epitopes were at least partially structural. However,some proteins (e.g. amylase) expressed predominantly primary sequenceepitopes. Typically, the epitopes were localised in very discrete areasof the enzymes, and different epitope sequences often shared some aminoacids (hot-spots).

The identified epitope sequences are shown in Tables 2-7.

Birch Allergen:

Bet v1 (WO 99/47680) was used as the parent protein for identificationof epitope sequences that may cross react with enzyme epitopes. Thestructural coordinates from 1BV1.pdb (Gajhede et al., NAT.STRUCT.BIOL.,Vol. 3, p. 1040, 1996) were used as well the corresponding sequence(Swissprot accession number P15494). The epitope pattern P>PAP>S (whichhad been identified from antibody binding peptides specific foranti-Lipolase antibodies) was found to match three (overlapping) epitopesequences on the surface of Bet v1:

Bet v1 1.1: P31 A34 P35 A37 P59 S39/S40; Bet v1 1.2: P63 L62 P59 A37 P35S39/S40; and Bet v1 1.3: P59 S39/S40 P31 A34 P35 S39/S40. Example 3Epitope Areas

It is common knowledge that amino acids that surround binding sequencescan affect binding of a ligand without participating actively in thebinding process. Based on this knowledge, areas covered by amino acidswith potential steric effects on the epitope-antibody interaction, weredefined around the identified epitopes. Practically, all amino acidssituated within 5 Å from the amino acids defining the epitope wereincluded. The accessibility criterium was not included for definingepitope areas, as hidden amino acids can have an effect on thesurrounding structures.

For Savinase, the following amino acid residues belong to the epitopearea that correspond to each epitope sequence indicated in Table 2:

sav1.1 A1 Q2 S3 P5 H39 P40 D41 L42 N43 G63 T66 H67 A69 G70 T71 A73 A74L75 N77 S78 I79 G80 V81 L82 G83 N204 V205 Q206 S207 T208 Y209 P210 S212T213 Y214 A215 S216 L217 sav1.2 S153 G154 N155 S156 G157 A158 G160 S161I162 S163 A169 R170 A174 M175 A176 V177 G178 R186 F189 S190 Q191 Y192G193 A194 G195 L196 D197 I198 V199 T220 R247 K251 A254 T255 S256 T260N261 L262 Y263 G264 S265 G266 L267 sav2.1 W6 G7 I8 R10 V11 Q12 A13 P14A15 A16 R19 L21 V84 T180 D181 Q182 N183 N184 I198 V199 A200 P201 H226V227 A230 L233 V234 K237 N238 H249 L250 T253 A254 T255 S256 L257 S265G266 L267 V268 N269 A270 E271 A272 A273 T274 R275 sav2.2 S153 G154 N155S156 G157 A158 S161 I162 S163 G178 A179 T180 D181 N184 N185 R186 A187S188 F189 S190 Q191 Y192 G193 L196 T220 L262 Y263 sav2.3 A142 T143 G146V147 L148 Y171 A172 N173 A174 M175 D197 A231 V234 K235 N238 P239 S240W241 S242 N243 V244 Q245 I246 R247 N248 H249 L250 K251 sav3.1 S153 G154N155 S156 G157 A158 V177 G178 A179 T180 D181 N184 N185 R186 A187 S188F189 S190 Q191 Y192 V199 A200 P201 G202 V203 N218 G219 T220 A223 L262Y263 sav3.2 L111 E112 G115 N116 M119 A138 V139 N140 S141 A142 S144 R145G146 V147 V149 N173 N243 sav4.0 Q2 H17 T22 G23 S24 G25 V26 K27 V28 V30I35 S37 T38 H39 P40 D41 L42 N43 I44 R45 G46 T66 A69 G70 T71 I72 A73 A74L75 N76 N77 I79 G80 V81 L82 G83 V84 A85 P86 S87 A88 E89 L90 Y91 A92 T208Y209 P210 S212 T213 Y214 sav5.1 A1 Q2 S3 V4 I35 S37 H39 P40 D41 L42 N43I44 T66 A69 G70 A73 A74 L75 N76 N77 S78 I79 G80 V81 L82 G83 P86 L90 T208Y214 sav5.2 V30 T33 G34 I35 S37 T38 L42 N43 I44 R45 G46 E54 S57 T58 Q59D60 G61 N62 G63 H64 G65 T66 H67 A69 L90 Y91 A92 K94 P210 sav5.3 V4 P5 W6G7 I8 S9 R10 V11 Q12 A13 P14 A15 A16 R19 N269 A270 E271 A272 A273 T274R275 sav5.4 A1 Q2 P40 D41 F50 L75 N77 S78 I79 G80 V81 V104 S105 S106I107 A108 Q109 G110 L111 E112 W113 A114 G115 N116 Q137 A138 S141 A142Y214 sav6.1 V139 N140 T143 L148 V149 A151 P168 A169 Y171 A172 N173 A174M175 A176 D197 I198 N243 V244 Q245 I246 R247 N248 H249 L250 K251 N252T253 A254 S265 sav6.2 Q2 G25 V26 K27 V28 A29 I35 S37 T38 H39 P40 D41 L42N43 I44 R45 G46 G47 Q59 T66 A69 G70 A73 A74 L75 N77 I79 G80 V81 L82 A88E89 L90 Y91 N117 G118 M119 H120 V121 S207 T208 Y209 P210 G211 S212 T213Y214 A215 sav7.1 K27 L31 I107 A108 Q109 G110 L111 E112 W113 A114 G115N116 N117 G118 M119 A122 L124 L135 Q137 A138 V139 S141 A142 R145 V149sav7.2 V104 I107 A108 L111 S132 A133 T134 L135 E136 Q137 A138 V139 N140S141 A142 T143 S144 R145 G146 V147 V149 Y167 P168 Y171 A172 N173 A174M175 N243 R247 sav9.1 L111 E112 A114 G115 N116 M119 H120 V121 A122 E136Q137 A138 V139 N140 S141 A142 T143 S144 R145 G146 V147 L148 V149 V150N173 M175 N243 I246 R247 L250 sav9.2 L126 G127 S128 P129 A152 S153 G154S161 I162 S163 Y167 P168 A169 R170 Y171 A172 A176 V177 G178 Q191 Y192G193 A194 G195 L196 D197 I198 V199 T260 N261 L262 Y263 G264 sav10.1 Q12A13 P14 A15 A16 H17 N18 R19 G20 L21 T22 N76 L82 G83 V84 A85 P86 L233V234 K237 N238 H249 L250 T253 N269 A270 E271 A272 A273 T274 R275 sav10.2V11 Q12 A13 P14 A15 A16 H17 N18 R19 G20 L21 T22 G23 L233 V234 Q236 K237N238 H249 L250 T253 A254 T255 L267 V268 N269 A270 E271 A272 A273 T274R275 sav10.3 L31 D32 H64 V68 V95 L96 I107 L111 A114 G115 N116 M119 V121A122 N123 L124 S125 L126 G127 S128 P129 V139 S141 A142 T143 S144 R145G146 V147 L148 V149 V150 A151 A152 S153 S163 Y167 P168 A169 N173 A174M175 A176 V177 T220 S221 M222 T224 P225 V227 A228 A231 N243 I246 R247L250 sav10.4 P131 S132 A133 L135 E136 V139 A151 A152 S153 G160 S161 I162S163 Y167 P168 A169 R170 Y171 A172 N173 A174 A176 Q191 Y192 G193 A194G195 L196 R247 S259 T260 N261 L262 Y263 G264 sav11.0 W6 G154 N155 S156G157 A179 T180 D181 Q182 N183 N184 N185 R186 A187 S188 F189 S190 Q191Y192 P201 G202 V203 N204 V205 L217 N218 G219 T220 L262 Y263 sav12.0 L31I107 A108 Q109 G110 L111 E112 W113 A114 G115 N116 N117 G118 A122 L124S132 A133 T134 L135 Q137 A138 V139 N140 S141 T143 R145 V149 A151 S163Y167 P168 A169 R170 Y171 N173 A174 sav13.0 Q2 S3 P5 T38 H39 P40 D41 L42N43 H67 G70 A73 A74 L75 N77 I79 G80 V81 L82 G83 V205 Q206 S207 T208 Y209S212 T213 Y214 A215 S216 L217 sav14.0 A16 H17 R19 G20 L21 T22 G23 S24G25 V26 K27 V28 A29 V30 I35 I44 R45 G46 G47 V84 A85 P86 S87 A88 E89 L90Y91 A92 V93 W113 N117 G118 M119 H120 V121 A232 L233 K235 Q236 K237 T274sav15.0 W6 R10 G154 N155 S156 G157 V177 G178 A179 T180 D181 Q182 N183N184 N185 R186 A187 S188 F189 S190 Q191 V199 A200 P201 G202 V203 N218G219 T220 A223 L257 Y263 L267 sav16.0 A13 A16 H17 G20 L21 T22 G23 S24G25 V26 V28 I72 A73 V84 A85 P86 S87 A88 E89 L90 H120 G229 A230 A231 A232L233 V234 K235 Q236 K237 N238 P239 S240 W241 I246 H249 L250 A270 A273T274 sav17.0 T22 G23 S24 G25 V26 K27 V28 A29 V30 L31 D32 I35 I44 R45 G46G47 A48 F50 S87 A88 E89 L90 Y91 A92 V93 K94 V95 G110 W113 N117 G118 M119H120 V121 A232 K235 Q236 sav18.1 W6 G7 I8 S9 R10 V11 Q12 A179 T180 D181Q182 N183 N184 N185 R186 A187 I198 V199 A200 P201 V203 H226 V227 A230H249 L250 K251 N252 T253 A254 T255 S256 L257 S265 G266 L267 V268 N269A270 sav18.2 A13 A16 H17 L21 T22 G23 V26 V28 V84 A85 A88 V121 L148 Y171A172 N173 V174 M175 A176 G195 L196 D197 I198 V199 V227 A228 G229 A230A231 A232 L233 V234 K235 Q236 K237 N238 W241 N243 V244 Q245 I246 R247N248 H249 L250 K251 N252 T253 A254 Y263 G264 S265 G266 V268 A270 A273T274 sav19.1 A16 H17 R19 G20 L21 T22 G23 S24 G25 V26 K27 V28 S87 A88 E89H120 V121 A232 L233 V234 K235 Q236 K237 N238 P239 T274 sav19.2 A1 Q2 S3V4 P5 D41 H64 H67 G70 T71 A74 L75 N77 S78 I79 G80 V81 L82 G83 G202 V203N204 V205 Q206 S207 T208 Y209 Y214 A215 S216 L217 N218 G219 M222

For PD498, the following amino acid residues belong to the epitope areathat correspond to each epitope sequence indicated in Table 3:

pd1.1 D105 A108 S109 G110 I111 R112 Y113 A114 A115 D116 Q117 N131 S132T133 T134 L135 K136 S137 A138 V139 D140 Y141 A142 W143 N144 K145 G146A147 pd1.2 C128 E129 A153 G154 N155 D156 N157 V158 S160 R161 T162 F163Q167 S170 G178 A179 I180 D181 D184 R185 K186 A187 S188 F189 S190 N191Y192 G193 T194 W195 V196 T220 T262 N263 pd1.3 F50 L104 D105 S106 I107A108 S109 G110 I111 R112 Y113 A114 A115 D116 Q117 T133 T134 L135 K136S137 A138 V139 D140 Y141 A142 W143 N144 K145 G146 A147 pd1.4 T28 *28aVA29 V30 D32 S33 G34 V35 Y37 *44aaV I45 K46 G47 Y48 D49 F50 I51 R53 D54N55 N56 P57 M58 D60 L61 K89 I90 L91 A92 V93 R94 V95 L96 D97 A98 Y113A114 Q117 A119 pd1.5 D32 S33 G34 K46 G47 Y48 D49 F50 I51 D52 R53 D54 N55P57 M58 L61 L91 A92 V93 R94 V95 L96 D97 A98 L104 D105 S106 I107 A108S109 G110 I111 R112 Y113 A114 A115 D116 Q117 G118 A119 T133 T134 L135K136 S137 A138 V139 D140 Y141 A142 pd2.1 V19 T21 I111 R112 Y113 A114 A1D116 Q117 G118 A119 L122 D140 Y141 A142 W143 N144 K145 G146 A147 V148L233 L234 A235 S236 Q237 G238 K239 N240 N243 V244 Q245 I246 R247 Q248A249 A273 V274 R275 Y276 pd2.2 S24 S25 T26 Q27 T28 *28aV L42 A43 R44*44aK *44aaV I45 D75 N77 D87 T88 K89 I90 L91 G118 A119 K120 V121 L122G146 A147 V148 A232 A235 S236 pd2.3 R22 G23 S24 S25 T26 Q27 T28 *28aVD87 T88 K89 I111 A115 G118 A119 K120 V121 L122 S137 A138 V139 D140 Y141A142 W143 N144 K145 G146 A147 V148 V149 V150 I175 A231 A232 A235 S236N243 I246 R247 pd2.4 W-6 S12 T13 P14 A15 A16 V19 T21 R22 G23 S24 Q27L230 A231 L233 L234 A235 S236 Q237 G238 K239 N240 N243 Q245 I246 S270N271 K272 A273 V274 R275 Y276 pd3.1 L31 K46 G47 Y48 F50 L91 V93 S103L104 D105 S106 I107 A108 S109 G110 I111 R112 Y113 A114 A115 D116 Q117G118 L122 L124 C130 S132 T133 T134 L135 K136 S137 A138 V139 D140 Y141A142 Q167 P168 Y171 P172 pd3.2 V19 T21 R22 G23 S24 Q27 K120 V121 V148L230 A231 A232 L233 L234 A235 S236 Q237 G238 K239 N240 N243 Q245 I246R247 Q248 A249 I250 Q252 T253 K272 A273 V274 R275 Y276 pd4.1 W-6 S12 T13P14 A15 A16 W17 D18 V19 T21 R22 G23 S24 M84 A85 P86 D87 T88 A142 W143G146 A147 V148 G229 L230 A231 A232 L233 L234 A235 S236 Q237 G238 K239N240 N243 V244 Q245 I246 R247 Q248 A249 I250 S270 N271 A273 V274 R275Y276 pd4.2 W-6 T13 A16 W17 V19 T21 R22 G23 S24 *44aK A73 A74 *75aT G83M84 A85 P86 D87 T88 A142 G146 G146 A147 V148 G229 L230 A231 A232 L233L234 A235 S236 Q237 G232 K239 N240 N243 V244 Q245 I246 R247 Q248 A249I250 S270 A273 V274 R275 Y276 pd4.3 T26 Q27 T28 *28aV A29 V30 L31 Y37*44aaV I45 K46 G47 Y48 D49 D52 R53 D54 N55 N56 P57 M58 V72 T88 K89 I90L91 A92 V93 Y113 A114 A115 Q117 G118 A119 K120 V121 L122 N123 A147 A228A232 pd4.4 K46 G47 F50 L91 V93 S103 L104 D105 S106 I107 A108 S109 G110I111 R112 Y113 A114 A115 D116 Q117 G118 C130 S132 T133 T134 L135 K136S137 A138 V139 D140 Y141 Q167 P168 A169 S170 Y171 P172 N173 A174 pd4.5T28 *28aV A29 V30 L31 V35 D36 Y37 N38 H39 L42 A43 *44aaV I45 K46 G47 Y48F50 N55 N56 P57 M58 K89 I90 L91 A92 V93 A108 S109 G110 I111 R112 Y113A114 A115 D116 Q117 G118 A119 L122 pd5.0 F50 S103 L104 D105 S106 I107A108 S109 G110 I111 R112 Y113 A114 A115 D116 Q117 T133 T134 L135 K136S137 A138 V139 D140 Y141 A142 pd6.1 Y4 Y6 G7 G63 H64 H67 V68 T71 N155A179 F189 P201 G202 V203 N204 I205 A206 S207 V209 G213 Y214 S215 Y216M217 S218 G219 T220 S221 M222 A223 S224 P225 H226 pd6.2 W-6 T13 A16 W17V19 T21 R22 G23 S24 S25 Q27 M84 A85 P86 D87 T88 G229 L230 A231 A232 L233L234 A235 S236 Q237 G238 S270 V274 pd7.0 R22 G23 S24 S25 Q27 T28 *28aVA29 V30 V35 D36 Y37 N38 H39 P40 D41 L42 A43 R44 *44aK *44aaV T66 A69 G70V72 A73 A74 D75 N77 A85 P86 D87 T88 K89 I90 L91 A119 V121 L122 N123 T208A228 A231 pd8.0 W-6 T13 A16 W17 T21 R22 G23 Q27 *44aK A73 A74 *75aT G83M84 A85 P86 D87 T88 K120 V121 I175 A176 V177 G178 V196 D197 V198 T199A200 V227 G229 L230 A231 A232 L233 L234 A235 S236 Q237 G238 K239 N240N243 Q245 I246 Q248 A249 I250 Q252 T253 A254 F264 Y265 G266 I268 pd9.0W-6 Y6 G7 P8 Q9 N10 T11 S12 T13 P14 A15 A16 W17 D18 V19 T21 M84 V139W143 V148 V149 A151 P168 A169 Y171 P172 N173 A174 I175 A176 D181 S182N183 D184 D197 P201 L230 L233 L234 K239 N240 N243 V244 Q245 I246 R247Q248 A249 I250 E251 Q252 T253 A254 K267 I268 N269 S270 N271 K272 A273V274 R275 Y276 pd10.0 L124 L126 G127 C128 E129 C130 N131 L135 V139 A151A152 A153 G154 N155 D156 N157 V158 S160 R161 T162 F163 Q167 P168 A169S170 Y171 A174 I175 A176 N191 Y192 G193 T194 W195 V196 T262 N263 F264*264aK pd11.0 W-6 S-5 Y2 Y4 Q5 Y6 G7 P8 Q9 N10 T11 S12 T13 P14 W17 D18V19 T21 A82 M84 I180 D181 S182 N183 D184 P201 G202 V203 N204 I205 H226L233 S270 N271 V274 R275 pd12.0 G127 C128 E129 V139 V148 V149 V150 A151A152 A153 G154 N155 D156 V158 R161 T162 F163 Q167 P168 A169 S170 Y171P172 N173 A174 I175 A176 V177 G178 N191 Y192 G193 T194 W195 V196 D197V198 T199 A200 V227 R247 I250 E251 A254 N263 F264 *264aK Y265 G266 I268pd13.1 W-6 S-5 P-4 D-2 P-1 Y1 Y2 S3 *3aA Y4 Q5 P8 Q9 S12 T13 P14 A15 A16W17 D18 V19 T21 R22 G80 V81 A82 N271 V274 R275 pd13.2 W-6 S-5 P-4 N-3D-2 P-1 Y1 Y2 S3 *3aA Y4 Q5 P8 Q9 P14 W17 D41 G70 A74 D75 *75aT N76 N77G78 I79 G80 V81 A82 G83 A206 S207 T208 Y214 pd14.0 T28 V35 D36 Y37 N38H39 P40 D41 L42 A43 R44 *44aK *44aaV I45 K46 G47 Y48 D49 F50 R53 D54 N55N56 P57 M58 T66 A69 G70 A73 A74 D75 K89 I90 L91 A92 V93 R94 Y113 T208pd15.0 V30 L31 D32 S33 G34 V35 D36 Y37 N38 H39 L42 A43 *44aaV K46 Y48D49 F50 I51 N56 P57 M58 D60 L61 N62 G63 H64 G65 T66 A69 I90 A92 V93 R94V95 L96 D97 A98 G100 S101 G102 S103 S106 I107 G110 S125 L126 V209 P210N211 N212 pd16.0 W-6 S-5 P-4 N-3 Y2 G7 P8 Q9 N10 T11 S12 T13 P14 A15 A16W17 D18 V19 T21 R22 *75aT N76 A82 G83 M84 A85 P86 L233 N269 S270 N271pd17.1 T11 S12 A15 A16 D18 V19 T21 R22 G23 S24 Q27 L230 A232 L233 L234A235 S236 Q237 G238 K239 N240 N243 Q245 I246 Q248 A249 Q252 T253 N269S270 N271 K272 A273 V274 R275 Y276 pd17.2 A108 I111 R112 A115 D116 K120L124 T133 T134 L135 K136 S137 A138 V139 D140 Y141 A142 W143 N144 K145G146 A147 V148 V149 P168 Y171 N173 A174 N243 pd18.1 W-6 T13 A16 W17 V19T21 R22 G23 S24 S25 *44aK M84 A85 P86 D87 T88 K89 G229 L230 A231 A232L233 L234 A235 S236 Q237 K239 A249 I250 T253 N269 S270 N271 K272 A273V274 R275 Y276 pd18.2 D-2 V30 V35 D36 Y37 N38 H39 P40 D41 L42 A43 R44*44aK *44aaV I45 K46 G47 Y48 P57 T66 A69 G70 A73 A74 D75 *75aT N76 N77I79 V81 A82 A85 P86 D87 T88 K89 I90 L91 A92 V93 R94 T208

For Lipolase, the following amino acid residues belong to the epitopearea that correspond to each epitope sequence indicated in Table 4:

lip2.1 Y53 F55 V63 L78 F80 W117 V120 A121 D122 T123 L124 R125 Q126 K127V128 E129 D130 A131 V132 R133 V140 L159 R160 G161 N162 G163 Y164 D165I166 G190 lip2.2 V2 L6 F10 A173 P174 R175 A182 L193 Y194 R195 I196 T197P204 R205 Y213 S214 H215 S216 S217 P218 E219 Y220 W221 I222 I235 V236K237 I238 E239 I241 D242 A243 G246 N247 N248 lip2.3 V2 L6 F10 A182 L185T186 L193 Y194 R195 I196 T197 H215 S216 S217 P218 E219 Y220 W221 I222I235 V236 K237 I238 E239 G240 I241 A243 G246 N247 N248 lip2.4 V2 L6 F10L193 Y194 R195 I196 T197 S216 S217 P218 E219 Y220 W221 I222 I235 V236K237 I238 E239 G240 A243 G246 N247 N248 lip3.0 L93 K94 F95 H110 A173P174 R175 V176 G177 N178 R179 A182 L185 T186 L193 R195 N200 D201 I202P204 R205 L206 P207 P208 R209 E210 F211 G212 Y213 S214 H215 S216 S217P218 E219 I238 E239 G240 I241 D242 A243 T244 G245 N248 ?R259? P250 N251I252 P253 D254 I255 lip4.0 R175 V176 G177 N178 R179 A180 F181 A182 E183F184 L185 T186 R205 P207 P208 R209 E210 F211 G212 Y213 S214 H215 S216S217 I241 D242 N248 lip5.1 A20 Y21 N25 N26 T50 F51 L52 Y53 S54 F55 E56V63 T64 G65 F66 L67 A68 L69 I76 V77 L78 S79 F80 R81 G82 S83 R84 S85 I86E87 N88 W89 K127 V128 A131 H145 S146 L147 G148 L151 G266 lip5.2 K94 F95L96 L97 K98 E99 R108 G109 H110 D111 G112 R175 V176 G177 N178 R179 A180F181 A182 E183 F184 R205 P207 P208 R209 E210 F211 G212 Y213 S214 H215S216 I241 D242 N248 lip6.0 Q9 F10 N11 F13 A14 S17 V63 F80 R81 W89 L93F113 S116 W117 F142 T143 G144 H145 S146 L147 G148 G149 A150 L151 A152T153 V154 A155 G156 A157 V168 F169 S170 Y171 G172 A173 P174 R175 V176F181 L185 L193 Y194 R195 I196 T197 D201 V203 P204 L206 P207 H215 H258Y261 F262 I265 lip7.0 F13 A14 Q15 Y16 S17 A180 A19 A20 Y21 C22 G23 N25N26 I34 C36 A40 C41 F51 L52 Y53 S54 F55 E56 V63 T64 G65 F66 L67 S79 F80R81 V120 A121 D122 T123 L124 R125 Q126 K127 V128 L264 I265 lip8.1 L12F13 A14 Q15 Y16 S17 A18 A19 A20 I34 V44 A49 T50 F51 L52 F66 L67 A68 L69D70 N71 T72 N73 K74 L75 I76 V77 S79 H135 P136 D137 Y138 R139 V140 V141T143 lip8.2 L12 F13 A14 Q15 Y16 S17 A18 A19 A20 I34 V44 A49 T50 F51 L52Y53 S54 F55 G65 F66 L67 A68 L69 D70 N73 L75 I76 V77 L78 S79 T123 L124R125 Q126 K127 V128 E129 D130 A131 T143 lip9.0 L6 F10 N25 N26 D27 A28A30 G31 T50 F51 L52 Y53 S54 F55 E56 G65 F66 L67 A68 L69 I76 T123 L124R125 Q126 K127 V128 E129 D130 A131 V132 R1333 E134 H135 P136 R139 V140V141 F142 G156 L159 R160 G161 N162 G163 Y164 D165 I166 D167 V168 F169S170 G190 G191 T192 L193 Y194 R195 I196 Y220 lip10.0 N11 L12 Q15 Y16 I34T35 C36 C41 P42 E43 V44 E45 K46 A47 D48 A49 D70 N71 T72 N73 K74 lip11.0F95 L96 L97 K98 E99 I100 N101 D102 C107 R108 G109 H110 D111 F113 T114S115 A150 T153 V154 A173 P174 R175 V176 G177 N178 R179 F181 V203 P204R205 L206 P207 P208 R209 F211 G212 Y213 S214 H215 G240 I241 D242 A243T244 N248 lip12.0 L96 L97 K98 E99 I100 N101 D102 C104 S105 G106 C107R108 G109 H110 T114 S115 V176 G177 N178 A180 F181 F184 lip13.0 N11 L12F13 A14 Q15 Y16 S17 A182 A19 A20 Y21 N26 I34 C36 A40 C41 P42 E43 V44 A49F55 E56 V63 T64 G65 F66 L67 A68 D70 N73 L75 I76 V77 L78 S79 F80 R81 G82S83 R84 W89 W117 L124 V128 V141 F142 T143 G144 H145 S146 L147 G148 G149A150 L151 A152 A155 lip14.0 Q9 F10 N11 F13 A14 S17 Y21 R81 G82 S83 R84S85 I86 E87 N88 W89 I90 G91 N92 L93 F113 T143 G144 H145 S146 L147 G149A150 T153 V168 F169 S170 Y171 A173 P174 R175 V176 L193 Y194 R195 I196T197 D201 V203 P204 L206 P207 H215 H258 Y261 F262 I265 G266 lip15.0 N11L12 F13 A14 Q15 Y16 S17 A18 A19 A20 Y21 C22 G23 K24 N25 N26 D27 A28 I34T35 C36 A40 C41 P42 E43 V44 E45 K46 A47 A49 F51 L52 Y53 S54 F55 E56 T64G65 F66 L67 S79 F80 R81 T123 L124 K127 L264 I265 lip16.0 A14 E87 I90H145 G172 I196 T197 H198 T199 N200 D201 I202 P204 R205 W221 I222 K223S224 G225 T226 G246 N247 N254 I252 P253 D254 I255 P256 A257 H258 L259W260 Y261 F262 G263 I265 lip17.0 E1 V2 F7 F10 G177 N178 R179 A180 F181A182 E183 F184 L185 T186 L193 R195 H198 T199 G212 S214 H215 S216 S217P218 E219 Y220 W221 I222 K223 S224 G225 T226 V228 P229 V230 T231 R232N233 D234 I235 V236 K237 I238 E239 G240 I241 D242 A243 T244 G245 G246I262 lip18.0 Q9 F13 Y16 T32 N33 I34 C41 P42 E43 V44 E45 K46 A47 D48 A49T50 F51 L52 L67 A68 L69 D70 N71 T72 N73 L75 I76 V128 V132 H135 P136 D137Y138 R139 V140 V141 F142 Y164 D165 I166 D167 F169 Y194

For Amylase, the following amino acid residues belong to the epitopearea that correspond to each epitope sequence indicated in Table 5:

je1.1 N2 G3 T4 R33 P346 Y349 I352 L353 T354 R355 P360 V362 D366 Y367M378 K379 A380 K381 I382 D383 P384 I385 L386 E387 A388 R389 Q390 N391F392 A393 Y394 I450 T451 je1.2 Y57 D58 Y60 D61 F65 N66 Q67 L104 G105G106 A107 D108 A109 T110 E111 A135 W136 T137 K138 F139 D140 F141 P142G143 R144 G145 N146 T147 Y148 S149 F151 K152 W153 R154 F158 je2.1 M6 Y8E10 W11 H12 D26 L30 R33 V325 D326 N327 H328 D329 S330 Q331 P332 G333E334 E337 F339 K345 Y349 V362 F363 Y364 G365 D366 Y367 Y368 G369 I370P371 T372 H373 S374 V375 P376 A377 M378 K379 I382 D383 L386 je2.2 L289L293 V314 P318 T323 F324 V325 D326 F339 K345 P346 L347 A348 Y349 A350L351 I352 L353 T354 R355 F356 Q357 G358 Y359 P360 S361 V362 F363 Y364G365 D366 Y367 Y368 G369 P376 A377 M378 K379 I382 I385 R389 Q397 je2.3N102 V116 E117 V118 P120 R123 D159 G160 V161 D162 W163 Q168 F169 Q170N171 R172 I173 Y174 K175 A182 W183 D184 V187 D188 N193 Y194 D195 Y196L197 M198 Y199 A200 D201 V202 H236 je2.4 T1 N2 T4 M6 Y8 D26 L30 R31 N32R33 G34 I35 V325 D326 F339 K345 Y349 L353 V362 F363 Y364 G365 D366 Y367Y368 G369 I370 P376 A377 M378 K379 I382 D383 P384 I385 L386 E387 A388R389 Q390 N391 F392 Y394 H417 je3.1 M6 Q7 Y8 F9 E10 L13 H19 W20 N21 R22L23 R24 D25 D26 A27 S28 N29 L30 R31 N32 R33 I385 W39 I40 P41 P42 A43 W44V52 G53 Y54 Y75 A87 L88 N91 V93 D98 V100 Y364 Y368 je3.2 Y8 F9 W11 H19W20 W39 I40 P41 P42 A43 W44 D51 V52 G53 Y54 G55 A56 Y75 D98 V99 V100M101 N102 H103 L104 D195 L197 M198 A200 D201 V202 R230 I231 D232 A233V234 K235 H236 I237 E262 H328 je3.3 Y8 F9 H19 W20 W39 I40 P41 P42 A43W44 K45 G46 T47 V52 G53 Y54 G55 A56 Y57 D58 L59 Q67 K68 Y75 D98 V100L104 G105 G106 A107 D108 A109 T110 E111 A135 W136 T137 K138 F139 D140F141 P142 je4.1 L23 D25 D26 A27 S28 N29 L30 R31 N32 R33 G34 I35 T36 I38A84 I85 H86 A87 L88 K89 N90 N91 G92 V93 Q94 V95 Q390 je4.2 A43 W44 K45L59 Y60 D61 L62 G63 E64 F65 V71 R72 T73 K74 Y75 G76 T77 R78 S79 Q80 L81E82 S83 Y148 W219 Y220 T223 L224

Example 4

Having identified ‘antibody binding peptide’ sequences and by consensusanalysis also “epitope patterns” (e.g. >DF>>K>), one can identifypotential epitope sequences on the 3-dimensional surface of a parentprotein (=acceptor protein) in a semi-automated manner using thefollowing method:

The anchor amino acid residues are transferred to a three dimensionalstructure of the protein of interest, by colouring D red, F white and Kblue. Any surface area having all three residues within a distance of 18Å, preferably 15 Å, more preferably 12 Å, is then claimed to be anepitope. The relevant distance can easily be measured using e.g.molecular graphics programs like Insight!! from Molecular SimulationsInc.

The residues in question should be surface exposed, meaning that theresidue should be more than 20% surface exposed, preferably more than50% surface exposed, more preferably 70% surface exposed. The percentage“surface accessible area” of an amino acid residue of the parent proteinis defined as the Connolly surface (ACC value) measured using the DSSPprogram to the relevant protein part of the structure, divided by theresidue total surface area and multiplied by 100. The DSSP program isdisclosed in W. Kabsch and C. Sander, BIOPOLYMERS 22 (1983) pp.2577-2637. The residue total surface areas of the 20 natural amino acidsare tabulated in Thomas E. Creighton, PROTEINS; Structure and MolecularPrinciples, W.H. Freeman and Company, NY, ISBN: 0-7167-1566-X (1984).

Substitutions of one or more residue (s) within 18 Å, preferably 15 Å,more preferably 12 Å, around the geometrical center of the residuesinvolved in the epitope, for a bigger or smaller residues, may destroythe epitope, and make the protein less antigenic.

Residues involved in epitope is 2, preferably 3 and more preferably 4

Example 5 Production, Selection, and Evaluation of Enzyme Variants withReduced Antiqenicity or Immunogenicity

Epitope sequences and hot-spots amino acids were mutated using standardtechniques know to the person skilled in the field (e.g. site-directedmutagenesis, error-prone PCR—see for example Sambrook et al. (1989),Molecular Cloning. A Laboratory Manual, Cold Spring Harbour, N Y).

In the examples shown below, variants were made by site-directedmutagenesis. Amino acid exchanges giving new epitopes or duplicatingexisting epitopes, according to the information collected in theepitope-database (See Example 1), were avoided in the mutagenesisprocess.

Enzyme variants were screened for reduced binding of antibodies raisedagainst the backbone enzyme. Antibody binding was assessed bycompetitive ELISA as described in the Methods section.

Variants with reduced antibody binding capacity were further evaluatedin the mouse SC animal model (See methods section).

The following variants showed reduced IgE and/or reduced IgG levels inthe mouse model:

Parent % IgG % IgE protein Mutations Target epitope sequences responseresponse Savinase D181N Sav11.0; Sav15.0 and Sav18.1. 50 19 Hot spotamino acid. Savinase R170L; Q206E Sav9.4; Sav10.4; Sav1.1; and 5 34Sav19.2 Savinase R170L, S57P Sav9.4; Sav10.4 45 12 Savinase R247ESav2.3, Sav6.1, Sav18.2 75 30 Hot spot amino acid. Savinase R247QSav2.3, Sav6.1, Sav18.2 17 20 Hot spot amino acid. Savinase R247HSav2.3, Sav6.1, Sav18.2 40 27 Hot spot amino acid. Savinase R247KSav2.3, Sav6.1, Sav18.2 74 34 Hot spot amino acid.

Example 6 Production, Selection, and Evaluation of Enzyme Variants withReduced Antigenicity or Immunogenicity

Hot-spots or epitopes were mutated using techniques known to the expertin the field (e.g. site-directed mutagenesis, error-prone PCR).

In the examples showed below, variants were made by site-directedmutagenesis. Amino acid exchanges giving new epitopes or duplicatingexisting epitopes according to the information collected in theepitope-database, were avoided in the mutagenesis process.

Enzyme variants were screened for reduced binding of antibodies raisedagainst the backbone enzyme. This antibody binding was assessed byestablished assays (e.g. competitive ELISA, agglutination assay).

Variants with reduced antibody binding capacity were further evaluatedin animal studies.

Mice were immunised subcutanuous weekly, for a period of 20 weeks, with50 microliters 0.9% (wt/vol) NaCl (control group), or 50 microliters0.9% (wt/vol) NaCl containing 10 micrograms of protein. Blood samples(100 microliters) were collected from the eye one week after everysecond immunization. Serum was obtained by blood clothing, andcentrifugation.

Specific IgG1 and IgE levels were determined using the ELISA specificfor mouse or rat IgG1 or IgE. Differences between data sets wereanalysed by using appropriate statistical methods.

A. Site-Directed Mutagenesis of Amino Acids Defining Epitopes, with anEffect on IgG1 and/or IgE Responses in Mice.

Epitope: A172/A169 R170A194 G193 N261 Pattern: AR>R>A>N Antibody:IgG1+IgE Backbone: Savinase

The variant carried the mutation R170F.

In a competitive IgE ELISA, this variant was less effective in competingfor anti-savinase antibodies, giving a 15% lower endpoint inhibition ascompared to the savinase backbone.

Mouse studies revealed an 80% reduction of the specific IgE levels, ascompared to savinase backbone (p<0.01). The IgG1 levels were notsignificantly affected.

Epitope: S216 E219 Y220 Pattern: E Y>M Antibody: IgG1 Backbone:Lipoprime

The variant carried the mutation S216W.

In a competitive IgG ELISA, the variant was less effective in competingfor Lipolase antibodies, giving a 38% decrease in endpoint inhibition ascompared to the enzyme backbone.

Mouse studies revealed a 69% decrease in specific IgG1 levels, comparedto the lipolase backbone (p<0.05). The IgE levels were not significantlyaffected.

B. Site-Directed Mutagenesis of Epitopes, with Examples of EpitopeDuplication, and New Epitope Formation, Respectively, Predicted by theEpitope-Database.

Epitope: T143 N173 N140 E136 L135 Pattern: S/T NN>EL Antibody: IgG1Backbone: Savinase

The variant carried the mutation E136R.

In a competitive IgG ELISA, the variants were less effective incompeting for savinase antibodies, giving a 38% decrease in endpointinhibition as compared to the savinase backbone.

Mouse studies revealed a dramatic increase in specific IgG1 levels,compared to savinase backbone (p<0.01). The IgE levels were notsignificantly affected.

Mutation E136R establishes an IgG1 epitope of the R Y P R/K pattern,previously identified on PD498. Apparently, this new epitope was moreantigenic in mice than the existing epitope. The introduction of asavinase unrelated epitope on the savinase backbone could explain theobserved discrepancy between competitive ELISA and animal studies.

In this example, it was found that using information derived exclusivelyfrom screening phage libraries with anti-PD498 antibodies (to identifythe R Y P R/K epitope pattern of Table 2) one could predict the outcomeof a genetic engineering experiment for Savinase in which the E136Rmutation created the PD498-epitope on the Savinase surface, leading toincreased immunogenicity of this Savinase variant. This demonstratesthat the epitope patterns identified may be used to predict the effecton immunogenicity of substitutions in proteins that are different fromthe parent protein(s) used to identify the epitope pattern.

C. Site-Directed Mutagenesis of Amino Acids Defining Epitope Areas, witha Differential Effect on IgG1 and IgE Antibody Levels in Mice, and anInhibiting Effect on IgG Binding, Respectively.

Epitope: A172/A169 R170A194 G193 N261 Pattern: AR>R>A>N Antibody:IgG1+IgE Backbone: Savinase

Epitope area: P131, S132, A133, L135, E136, V139, A151, A152, S153,G161, S162, I165, S166, Y167, P168, Y171, N173, A174, A176, Q191, Y192,G195, L196, R247, S259, T260, L262, Y263, G264.

The variant was different at position Y167 by the mutation Y167I.

In a competitive IgE ELISA, the variant was less effective in competingfor anti-savinase antibodies, giving a 8% lower endpoint inhibition ascompared to the its backbone.

Mouse studies revealed a 75% reduction of the specific IgE levels, ascompared to the backbone (p<0.01). In contrast, the IgG1 levels weredramatically increased (p<0.01).

Epitope: T143 N173 N140 E136 L135 Pattern: S/T NN>EL Antibody: IgG1Backbone: Savinase

Epitope area: V10A, 1107, A108, L111, E112, G115, S132, A133, T134,Q137, A138, V139, S141, A142, S144, R145, G146, V147, V149, Y167, P168,Y171, A172, A174, M175, N243, R247.

While variant no. 1 was mutated at the epitope position (N140D), variantno. 2 was mutated at N140 (N140D), but also at the epitope area position(A172D).

In a competitive IgG ELISA, variant no. 1 was less effective incompeting for anti-savinase antibodies, as compared to savinase. Thisvariant revealed a 21% lower endpoint inhibition as compared to the itsbackbone.

Variant no. 2 resulted in an endpoint inhibition that was 60% lower ascompared to savinase, and 40% as compared to variant no. 1.

Example 7 Conjugation of Savinase Variant E136K with ActivatedBis-PEG-1000

4.9 mg of the Savinase variant was incubated in 50 mM Sodium Borate pH9.5 with 12 mg of N-succinimidyl carbonate activated bis-PEG 1000 in areaction volume of approximately 2 ml. The reaction was carried out atambient temperature using magnetic stirring while keeping the pH withinthe interval 9.0-9.5 by addition of 0.5 M NaOH. The reaction time was 2hours.

The derivatives was purified and reagent excess removed by sizeexclusion chromatography on a Superdex-75 column (Pharmacia)equilibrated in 50 mM Sodium Borate, 5 mM Succinic Acid, 150 mM NaCl, 1mM CaCl₂ pH 6.0.

The conjugate was stored at −20° C., in the above described buffer.

Compared to the parent enzyme variant, the protease activity of theconjugate was retained (97% using Dimethyl-casein as substrate at pH 9).

Example 8

Competitive ELISA was performed according to established procedures. Inshort, a 96 well ELISA plate was coated with the parent protein. Afterproper blocking and washing, the coated antigen was incubated withrabbit anti-enzyme polyclonal antiserum in the presence of variousamounts of modified protein (the competitior).

The amount of residual rabbit antiserum was detected by pig anti-rabbitimmunoglobulin, horseraddish peroxidase labelled.

Epitope: T143 N173 N140 E136 L135 Pattern: S/T N N > E L Antibody: IgG1Backbone: Savinase Mutation: E136K Modification: bis-NHS-PEG1000

The data show that the derivative (60% endpoint inhibition) has reducedcapacity to bind enzyme specific immunoglobulines, as compared to theparent protein (100% endpoint inhibition).

Example 9

For this example the epitope sequences were determined in fourenvironmental allergens (Bet v1; Der f2; Der p2 and PhI p2), based ontheir structures (1btv.pdb; 1ahm.pdb; a19v.pdb; and 1whp.pdb,respectively), sequences (SEQ ID NOS: 6, 7, 8 and 9, respectively) andcomputer modelling of the epitope patterns that had been assembled inour database (shown in Table 8). The allergens arise from common sourcesof allergy: Birch (Bet v1 from Betula pendula), House dust mites (Der f2from Dermatophagoides farinae and Der p2 from Dermatophagoidespteronyssinus), and Timothy grass (PhI p2 from Phleum pratense).

The protein surface is scanned for epitope patterns matching the given“consensus” sequence of about 6-12 residues. First, residues on theprotein surface that match the first residue of the consensus sequenceare identified. Within a specified distance from each of these, residueson the protein surface that match the next residue of the consensussequence are identified. This procedure is repeated for the remainingresidues of the consensus sequence. The method is further describedunder the paragraph “Methods” above and the computer program can befound in the Appendixes.

The critical parameters used in this screening included:

-   -   i) a maximal distance between the alpha-carbon atoms of        subsequent amino acids,    -   ii) a minimal accessibility of the amino acid of 20 Å2,    -   iii) the largest maximal distance between the most distinct        amino acids should be less than 25 Å,    -   iv) the 5 best epitopes were taken,    -   v) the minimal homology with the epitope pattern of interest was        80%

In this way a number of potential epitopes are identified. The epitopesare sorted according to total surface accessible area, and certainentries removed:

-   -   1) Epitopes that contain the same protein surface residue more        than once. These are artefacts generated by the described        algorithm.    -   2) Epitopes which are “too big”, i.e. where a distance between        any two residues in the epitope exceeds a given threshold.

The epitope sequences found by this second generation mapping procedurewere:

Bet v1: Epi#02 A146, K32, Q36, F30, T142, R145, V12 A34, K32, Q36, F30,T142, R145, V12 Epi#03 L62, K65, - - - , 156, Y66 L24, K20, H76, I23,Y81 L24, K20, H76, 1104, Y81 Epi#04 K134, S136, Q132, K129, A130, A135K134, S136, Q132, K129, V128, G1 Epi#05 G140, A146, R145, T10, G111,A106, T107, V12 G26, A146, R145, T10, G110, A106, T107, V12 G140, A146,R145, T10, G110, S11, S149, L152 G110, A106, S11, T9, G140, R145, T10,V12 G140, A146, R145, T10, G111, S11, S149, V12 Epi#06 G110, P108, D109,T107, A106, P14 G111, P108, D109, T107, A106, P14 A34, N28, D27, S40,K32, P35 G26, N28, D27, S39, K32, P35 A106, N78, D75, T77, A16, P14 G26,N28, D27, S39, Q36, P35 Epi#07 G46, T52, D69, S99, R70, V71, P50, D72G49, T52, D69, S99, R70, V71, P50, D72 G48, T52, D69, S99, R70, V71,P50, D72 Epi#08 K123, E127, G1, V2, H121, F3 K65, E60, F64, V67, F58K65, E60, F58, V67, F64 K129, E127, G1, V2, H121, F3 Epi#09 S149, L152,D156, N159, R17, L24, D75, K103, N78, A106, V12 L152, S149, D156, N159,R17, L24, D75, H76, N78, A106, V12 L152, D156, N159, R17, L24, D75, K80,N78, A106, V12 Epi#10 D109, A106, N78, T77, F79, R17, K20 E141, T10,R145, T142, F30, G26, K32 E8, T10, R145, T142, F30, G26, K32 Epi#11 F30,K32, I38, Q36, V33, E148 F22, F30, I38, Q36, V33, E148 F30, L143, 138,Q36, V33, E148 Epi#12 Y5, E6 Y83, E73 Y120, E127 Y5, E8 Y66, E87 Y81,E73 Epi#13 H76, A16, P14, T107, A106, P108, G110, G111 A16, R17, P14,T107, A106, P108, G110, G111 A157, R17, P14, T107, A106, P108, G111,G110 Epi#15 K65, P90, D93, I91, K97, G92 K32, P31, D27, I56, K65, G61Epi#17 A153, S149, R145, S11 A106, S11, R145, S149 Epi#18 R145, S149,L152, A153, Y150, L151, H154, S155 R145, S149, L152, A153, S155, L151,A157, N159 Epi#22 D125, D93, P90, K65 D93, P90, P63, E60 Epi#23 K55,N43, E42, S57, L62, P63 K68, N43, E42, S40, F30, P35 K54, N43, E42, S57,F64, P63 K55, N43, E42, S40, F58, P35 Epi#24 E96, K97, E87, P90, F64,E60, K65 E127, K123, E96, P90, F64, P63, K65 E42, K68, E87, P90, F64,E60, K65 E42, K55, E87, P90, F64, E60, K65 D93, G92, E87, P90, F64, E60,K65 D125, K123, E96, P90, F64, P63, K65 Epi#25 R70, K55, I44, E45, E42R70, K54, I44, E45, N47 R70, K68, I53, E45, N47 Epi#27 D93, E127, D125,K123 Epi#28 A146, Q36, F58, E60, L62, F64, P63, K65 I38, Q36, F58, E60,L62, F64, P63, K65 A34, Q36, F58, E60, L62, F64, P63, K65 L143, Q36,F58, E60, L62, F64, P63, K65 V33, Q36, F58, E60, L62, F64, G61, K65Epi#29 G61, K65, L62, F58, E60 I56, K65, L62, F64, E60 G89, K65, L62,F64, E60 V67, K65, L62, F64, E60 Epi#30 G1, N4, S99, H121, K97, I91, P90I113, I13, S149, H154, S155, L152, L151 I13, L152, A153, H154, S155,L151, V33 G110, I13, S149, H154, S155, L152, L151 G1, N4, S99, H121,K97, I98, V2 G1, N4, S99, H121, K97, I91, V85 Epi#33 K32, F30, P35, S39,S57, K65 Q36, F30, P35, S39, S40, K32 K32, F30, P35, S40, S57, K65 K65,F58, P35, S39, A34, R145 Epi#34 V105, P14, T107, V12, R145, Y150, S155I113, P14, T107, V12, R145, Y150, S155 Epi#37 P50, V74, L24, R17, N159P50, V74, L24, K20, N159 P14, R17, L24, K20, N159 Epi#38 L143, G140,E141, R145, V33, N28, P31, S39 L143, G140, E141, R145, V33, N28, P31,S40 L143, G140, E141, R145, V33, N28, P31, S57 Epi#39 A130, E127, H126,T94, P90, G89, L62 A130, E127, H121, T94, P90, G89, L62 Epi#40 A157,L152, A153, Y150, K32, S39 A153, L152, A157, Y150, K32, S40 R17, L151,A153, Y150, K32, S40 R145, L143, A34, Y150, A153, S155 R145, L143, G140,T9, K115, T10 Epi#41 P63, Y66, L62, S57 Epi#44 I23, R17, D156, Y150,S149, V12, T10 L24, R17, D156, Y150, S149, V12, P14 L24, R17, D156,Y158, A16, A106, P108 I13, R17, D156, Y150, S149, V12, T10 L151, R17,D156, Y150, S149, V12, T10 L24, R17, D156, Y150, S149, V12, T107 Epi#45K32, P35, F30, Y150, R145, M139, G140 K32, P35, F30, Y150, R145, M139,L143 K32, P31, F30, Y150, R145, M139, G140 Epi#47 L152, S149, R145,L143, A34, F30, N28, P31, P35 A153, S149, R145, A146, A34, F30, N28,P31, P35 Epi#48 E60, K65, P90, P63, G61 E60, K65, P63, P90, G92 Epi#51T94, H126, E127, D125, G124, K123, H121 D125, H126, E127, T94, K123,T122, H121 Der f2: Epi#02 A98, K100, S101, P99, R128, R31 A98, K100,R128, P99, R31, V94 T91, N93, P95, P34, R31, R128 L61, N93, P95, P34,R31, R128 Epi#03 L40, K15, A39, I13, Y86 L40, K14, A39, I88, Y90 Epi#05G32, A98, R31, P34, G20, T36, T91, Y90 G32, A98, R31, P34, G20, T36,T91, V94 G32, A98, R31, P34, G20, T36, T91, L37 G32, A98, R31, P34, G20,T36, T91, V18 Epi#06 A98, P99, D129, R31, K96, P95 G32, P99, D129, R128,R31, P95 A98, P99, D129, R31, K33, P95 A98, P99, D129, R31, K96, P34A98, P99, D129, R128, K126, P26 Epi#07 T107, S57, D59, S101, R128, A98,P99, D129 T107, S57, D59, S101, R31, A98, P99, D129 Epi#08 K15, D87,V76, H74, F75 K14, D87, V76, H74, F75 K77, D87, V76, H74, F75 Epi#09L61, D64, I68, H74, F75, T70, N71 N114, N46, D113, K48, N71, T70, T49G83, N46, D113, K48, N71, T70, T49 Epi#10 L40, I13, D42, N44, V81, K48,N46, N114, G115 L40, I13, D42, N44, V81, K82, N46, N114, G115 L37, D19,G20, V18, V3, D4, K6, A120, T107, V105 Epi#11 F75, K51, I111, Q45, V116,D113 F75, K51, I111, Q45, V81, D113 Epi#12 Y90, E38 Epi#13 H30, R31,P95, A98, P99, S101, G60, L61 Epi#15

K96, P99, D129, 128, R128, A98

K96, P99, D129, I127, R128, A98 K96, P99, D129, I29, R128, A98 K55, P66,D64, I68, T70, G67 Epi#18 R31, R128, I28, G125, T123, H124, V105 R31,R128, I127, G125, T123, H124, V105 Epi#22 D1, M17, D4, V3, K6 D1, M17,D19, P34, K96 D1, M17, D4, V5, K6 Epi#23 K14, N11, E12, N44, Q85, P79K14, N11, E12, N10, Q45, P79 K14, N11, E12, N44, Q84, P79 K14, N11, E12,L40, Q85, P79 Epi#24 D129, K100, E102, P99, R128, R31, K96 E62, G60,E102, P99, R128, R31, K96 D129, K126, E102, P99, R128, R31, K33 D129,K126, E102, P99, R31, P95, K96 Epi#25 R31, K96, I97, D59, E62 R128, R31,I97, D59, E102 R128, K126, I127, E102, N103 Epi#27 D64, E62, D59, K100D59, E62, D64, K55 D87, E38, D19, K33 D19, E38, D87, K15 D19, E38, D87,K14 D19, E38, D87, K77 Epi#28 V16, D87, Q85, K14, E12, K15, Q2, D1 I13,D87, Q85, K14, E12, K15, Q2, D1 V3, D1, Q2, K15, E12, K14, Q85, D87 L40,D87, Q85, K14, E12, K15, Q2, D1 I88, D87, Q85, K14, E12, K15, Q2, D1V76, D87, Q85, K14, E12, K15, Q2, D1 V18, D1, Q2, K15, E12, K14, Q85,D87 Epi#29 G32, N93, L61, E62 V94, N93, L61, E62 Epi#30 G60, I97, A98,H30, K96, P34, P95 I68, N71, H74, K77, P79, V81 G32, I97, A98, H30, K96,P95, P34 Epi#34 V105, P26, S24, G125, R128, S101, P99 W92, P34, T91,V94, R31, S101, P99 I28, P26, T123, G125, R128, S101, P99 Epi#37 A120,V16, L40, K14, N11 A39, V16, L40, K14, N11 Y90, A39, L40, K14, N11 Y86,A39, L40, K14, N11 Epi#39 A120, E38, T91, P34, G20, L37 A39, E38, T91,P34, G20, L37 Epi#40 G20, L37, A120, T123, K6, S24 A39, L37, A120, T123,K6, S24 G20, L37, A120, T107, K6, T123 Epi#41 P34, L37, V106, S57 Epi#42P26, S24, G125, R128, R31 P99, S101, G125, R128, R31 Epi#44 V16, Q2,D19, P34, W92, Y90, A39, V18, T91 V16, Q2, D19, P34, W92, Y90, A39, V5,T123 V3, Q2, D19, P34, W92, Y90, A39, V18, T91 Epi#45 K77, H74, F75,N71, D69, G67

K77, H74, F75, N71, D69, V76

K77, H74, F75, N71, D69, V65 Epi#46 A98, R128, R31, P95, N93, G32 A98,R128, R31, P34, G20, Q2 Epi#48 Q2, D19, P34, P95, G32 H30, K96, P95,P34, G20 Epi#49 D87, D42, L40, Q85, Q84, C78, T47, Q45, K48 D87, D42,L40, Q85, Q84, C78, T47, Q45, K82 Epi#50 D19, W92, P34, T91 D19, W92,P34, P95 D19, W92, T91, T36 Epi#51 D129, H30, K33, R31, R128, K126, H124R31, H30, D129, R128, K100, K126, H124 T123, H124, K126, R128, R31, K33,H30 Der p2: Epi#03 L17, K89, A39, I13, Y86 L17, K89, A72, I88, Y90 L17,K89, A72, I52, Y90 Epi#04 K15, S1, Q2, K14, V16, L17

K15, S1, Q2, K14, A39, L17

K15, S1, Q2, K14, V40, I13 Epi#05 G60, A56, L61, P99, G32, R31, H30, I97G60, A56, L61, P99, G32, R31, H30, I28 Epi#06 G60, A56, D64, S57, K55,P66 G83, N46, D114, T49, K48, P79 G60, N103, D59, S101, R31, P95 Epi#08K55, D64, S57, V106, F35 K55, E62, S57, V106, F35 Epi#09 L61, G60, E102,R128, I28, K126, N103, T123, V105 L61, G60, E102, R128, I127, K100,N103, T123, V105 L61, G60, E102, R128, I127, H124, N103, T123, V105Epi#10 SAS: 435, Size 24.47: D69, T91, N93, F35, G32, R31 SAS: 422, Size20.74: E38, T91, N93, F35, G32, K96 Epi#11 K14, I13, Q85, V81, E42 K15,I13, Q85, V81, E42 K14, I13, Q85, V40, D87 Epi#12 Y86, E42 Y90, E53 Y90,E38 Epi#13 H30, A125, P26, T123, A122, P19, L37, P34, W92 H30, A125,P26, T123, A122, H124, S24, G23, G20 H30, A125, P26, T123, A122, P19,L17, G20, F35 Epi#15 K55, P66, D69, I68, K89, A72 K55, P66, D69, I68,K89, A39 K55, P66, D64, I54, K109, G115 K55, P66, D64, I54, K109, A9Epi#18 R31, I29, A125, S101, E102, N103 R31, I29, A125, S101, E102, V104R31, I29, A125, T123, A122, V105 Epi#22 D69, P66, D64, V65, K55 D64,P66, D69, T91, K89 D59, L61, D64, P66, W92 D59, L61, D64, V65, E62 D69,P66, D64, V65, E53 Epi#24 D64, K55, E62, P99, R31, P34, K96 E53, K55,E62, P99, R31, P95, K96 D64, K55, E62, P99, R31, A98, K96 Epi#25 R31,H30, I28, E102, N103 R128, K126, I127, E102, N103 R128, K126, I28, E102,V105 Epi#27 D64, E53, D69, K89 D69, E53, D64, K55 D59, E62, D64, K55Epi#28 V40, D87, Q85, E42, Q84, G83, K82 G20, H22, Q2, L17, E38, L37,Q36, P34, K33 G20, H22, Q2, L17, E38, L37, F35, P34, K33 Epi#29 I97,K100, L61, E62 G60, N103, L61, E62 I127, N103, L61, E62 Epi#30 G60,N103, S101, H30, K96, I97, P95 G60, N103, A125, H30, K96, I97, P95 I28,I127, A125, H30, K96, I97, P95 Epi#33 Q36, F35, V106, S57, A56, K55 K33,F35, V106, S57, A56, K55 Epi#34 I28, P26, S24, G23, G20, T123, S57 I28,P26, S24, V3, G20, T123, T107 W92, P34, T91, V18, G20, T123, P26 Epi#37P66, V63, L61, K100, N103 P95, A98, L61, K100, N103 P19, V18, L17, K89,D87 P19, V3, L17, K89, D87 T123, V104, L61, K100, N103 Epi#38 L61, G60,E102, A125, V105, N103, P99, S57 L61, G60, E62, A56, V105, N103, P99,S57 Epi#39 A125, E102, H124, T123, P26, G20, L17 Epi#40 G60, L61, A56,T107, K6, T123 A39, L17, G20, T123, P26, S24 G60, L61, A56, T107, K55,S57 G60, L61, A56, T123, K126, S101 Epi#41 P19, L17, V3, S1 P19, L17,V5, S24 Epi#44 V65, D64, P66, W92, Y90, A39, V18, P19 L61, D64, P66,W92, Y90, A39, V18, T91 Epi#45 R31, P34, F35, N93, V94 K96, P34, F35,N93, G32 Epi#47 I127, S101, R31, I97, A98, L61, N103, P99, P95 I28,S101, R31, I97, A98, L61, N103, P99, S57 Epi#48 H30, K96, P95, P99, G60H30, K96, P34, P19, G20 H30, K96, P34, P19, V18 H30, K96, P34, P95, V94H30, K96, P34, P19, V3 E38, K89, P70, P66, V65 H30, K96, P95, P34, G32Q36, K89, P70, P66, V65 Epi#50 D69, Y90, W92, P66, P70 D69, Y90, W92,P34, P95 D69, Y90, W92, T91, P34 D69, Y90, W92, V94, P95 D69, Y90, W92,L37, P19 Epi#51 K126, H124, E102, R128, I28, R31, H30 T123, H124, K126,R128, I28, R31, H30 D4, H124, K126, R128, I28, R31, H30 PhI p2: Epi#02T87, K85, Q61, S38, R34, R67 T87, K85, Q61, P63, R34, V42 Epi#03 K10,A90, I88, Y86 K10, A18, I88, Y86 Epi#04 R34, S38, Q61, K85, T87, I88R34, S38, Q61, K85, T87, A90 Epi#05 G47, A18, S12, T87, G89, T91, T5, V1G73, A29, L69, T27, G50, T53, T45, V42 G11, A18, L20, T91, G89, A90,T87, I88 Epi#06 A93, P94, D79, R34, Q61, P59 A93, P94, D79, R34, Q61,P83 A93, P94, D80, R34, Q61, P59 A93, P94, D79, R34, Q61, P63 Epi#08K10, E9, G11, A18, H16, F54 K46, E48, G47, A18, H16, F54 K10, E9, S12,A18, H16, F54 Epi#09 L69, T27, G73, N76, R67, V77, D79, R34, A43, T45,V42 L69, T27, A29, E30, R67, V77, D80, R34, A43, T45, V42 Epi#10 D55,A18, N13, S12, F54, G47, K46 T45, A18, N13, S56, F54, G47, K46 Epi#09L60, S56, E57, D55, K15, N13, S12, G11 L60, S56, E57, D55, H16, F54,T45, T53 L60, S56, E57, D55, H16, F54, T45, G47 Epi#12 Y86, E84 Y23, E24Epi#18 N76, R67, F78, V81, A93, Y92, T91, T5, P2, V1 Epi#19 D39, W41,S38, Q61, R34, G37 E40, W41, S38, Q61, R34, A43 Epi#22 D79, P94, D80,P83, K85 D79, P94, D80, P63, K85 Epi#23 K10, N13, E14, L60, Q61, P59K10, N13, E14, L60, Q61, P83 K10, N13, E14, L60, Q61, P63 Epi#24 E58,K15, E57, P59, S56, E14, Q61 D55, K15, E57, P59, S56, E58, Q61 Epi#25R34, R67, W41, D39, E40 Epi#26 S38, E40, W41, V42, E32, E30 S38, E40,W41, V42, A43, E32 Epi#27 E14, E57, E58, K15 D55, E14, E84, K85 Epi#28G37, H36, Q61, K85, E84, L60, F54, A43, K46 G37, H36, Q61, K85, E84,L60, F54, S12, D55 G37, H36, Q61, K85, E84, L60, F54, S56, D55 G37, H36,Q61, K85, E84, L60, F54, A43, R67 G37, H36, Q61, K15, E57, L60, F54,A43, K46 G37, H36, Q61, K85, E84, L60, F54, S12, K15 G37, H36, Q61, K85,E84, L60, F54, S56, K15 G37, H36, Q61, K85, E84, L60, F54, A43, R34 G37,H36, Q61, K85, E84, L60, F54, A18, D55 Epi#29 G73, K72, L69, R67, E30I88, N13, L60, F54, E57 G25, K72, L69, R67, E32 V77, K75, L69, R67, E30G37, H36, L60, F54, E57 G37, Q61, L60, F54, E57 Epi#30 I88, N13, S12,H16, K15, P59, L60 I88, N13, S56, H16, K15, L60, P59 I88, N13, A18, H16,K15, P59, L60 Epi#33 K46, F54, V42, S56, K15 H16, F54, V42, S56, K15Epi#34 V1, P2, T5, V4, P94, Y92, T87 V1, P2, T5, L20, G89, T91, T87 V81,P94, T5, V1, P2, Y92, T91 Epi#37 T27, A29, L69, K72, D26 A43, R67, L69,K75, N76 Epi#38 L20, G89, E9, A18, N13, P59, S56 Epi#40 G49, L20, G89,Y86, K85, T87 G49, L20, G89, T87, K10, S12 G49, L20, G89, T87, K10, T7Epi#44 V77, R67, D79, P94, Y92, A93, V1, P2 L69, R67, D79, P94, Y92,A93, V1, T5 Epi#45 D79, P94, F78, N76, M74, L69 D80, P94, F78, R67, D79,V77 K3, P94, F78, N76, M74, G73 Epi#46 A43, R67, R34, P63, H36, Q61 V77,R67, R34, P63, H36, G37 L69, R67, R34, P63, G37, Q61 Epi#47 G37, E35,E40, A43, R34, L60, N13, P59, S56 V77, E32, E40, A43, R34, L60, N13,P59, S56 S38, G37, E40, A43, R34, L60, N13, P59, S56 Epi#48 E24, K3,P94, P2, V1 E84, D80, P94, P2, V1 Epi#50 D39, W41, A43, T45 D39, W41,V42, T45 Epi#51 D79, H36, E84, T87, K10, G11, H16 D39, H36, Q61, K85,P63, R34, W41 D79, H36, E40, D39, G37, R34, W41 Q61, H36, E84, T87, K10,G11, H16

TABLE 8 Each row indicates an epitope pattern. At each position (from 1to maximum of 12) the cells indicate which amino acids (single lettercoding) are allowed at that position. The last column indicates thepatterns identified using IgE antibody binding. Epitope Pattern PositionNumber 1 2 3 4 5 6 7 8 9 10 11 12 1 TS RQ YS NHC KR KR P HNP L IgE 2 RVR Y- PST FR- ALPQS- RKN ALT IgE (SEQ ID NO: 197) 3 Y I AH- K L 4 AGILANRTV- KRY Q S Y- KR (SEQ ID (SEQ ID NO: 198) NO: 199) 5 GILVY STH ASTR-G PT- RNAFLS A G IgE (SEQ ID (SEQ ID (SEQ ID NO: 200) NO: 201) NO: 202)6 P KRQSA STRC D PAN GA IgE (SEQ ID (SEQ ID NO: 203) NO: 204) 7 D P AV-R S- D S- T G 8 F HI- VA- FSG- DE- KA IgE 9 NRGLTV- STAN ANF RKH D-AILV- R- (ENRSV- AGI- DGNT- LIS- IgE (SEQ ID (SEQ ID (SEQ ID SEQ ID (SEQID NO: 205) NO: 206) NO: 207) NO: 208) NO: 209) 10 KR RG F C- AST- RNNTA DECT IgE (SEQ ID NO: 210) 11 DE V- Q I FLK F 12 E Y IgE 13 FWYGL PGALS- PH A T- P LRWA SAH IgE (SEQ ID (SEQ ID NO: 211) NO: 212) 14 GV QILV I- Y GNR DN TEH 15 AG RKQT- I D P RKN IgE (SEQ ID NO: 213) 16 DN ADA SDN QRSW GMR Y P RQL (SEQ ID NO: 214) 17 S- R S A 18 VLSFN AEHNPT- T-L- ST- Y- GAL LIV- CSF- R FRN- SD IgE (SEQ ID (SEQ ID NO: 215) NO: 216)19 AGLKM R Q QSC NTW DEI IgE (SEQ ID NO: 217) 20 D G D KN L LF- P K V AIgE 21 P S I- I LR- CI IgE 22 EDKW ACLPTV D- ASLPM- D- IgE (SEQ ID WY-(SEQ ID NO: 218) (SEQ ID NO: 220) NO: 219) 23 AP LQF SYLN- E N RK IgE(SEQ ID NO: 221) 24 KQ AELFPR- TSFR P EA GK DE IgE (SEQ ID (SEQ ID NO:222) NO: 223) 25 ENV DE IW- RKH R IgE 26 DE AGE PHV W E- S W IgE 27 K DEE DE IgE 28 DKR APSG- QF- CFIKLW- E FIKLW- Q DH- AGILV IgE (SEQ ID (SEQID (SEQ ID (SEQ ID NO: 224) NO: 225) NO: 226) NO: 227) 29 E RF- L KRQHNGGILV IgE P (SEQ (SEQ ID ID NO: NO: 229) 228) 30 LVP LIP IKLPQS- H AS-LIMN GI (SEQ ID (SEQ ID NO: 230) NO: 231) 31 D FI- MV- FW R N QR L 32 Vf- DE A A F 33 KR SA- S VP YF KQH IgE 34 STP STY GPR- GLV STM WP IVW 35I M S A- L AG 36 AW A PV- K- Q- ST Y- G- V- A A TP IgE 37 NYD KR L ARVTYAP IgE (SEQ ID NO: 232) 38 S P N LR- RV- AR- E G L 39 L G P RT- HL- EA 40 ST- APK- YT AG L- AGR IgE 41 St V- L Yh- P- 42 RQ R P- H- NQG S P L43 T- RI ML S HQ GL YA WC I 44 PT AGV SA Y W- P- D- RQ- ILVS IgE (SEQ IDNO: 233) 45 LVG MD- RN Y- F PH KRD IgE 46 AGQ HNQGC P R R AVLCY IgE (SEQID (SEQ ID NO: 234) NO: 235) 47 PS RP N LFQA- AR AILMNV RE AGSYLE LIAGVS(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 236) NO: 237) NO: 238) NO: 239) 48GV P P KHQD SHQE (SEQ ID (SEQ ID NO: 240) NO: 241) 49 KN Q- TMC WYC- Q QFP- VP- L W- D 50 PST STAPLW W WY- RHD V (SEQ ID NO: 242) 51 WH TSKHRQLIRKGP DSRTQG DEKQHT H RKQDT G (SEQ (SEQ ID KH- (SEQ (SEQ ID (SEQ ID IDNO: NO: 244) ID NO: NO: 246) NO: 247) 243) 245) 52 Q DNT- W R STRE- A FW(SEQ ID NO: 248)

Example 10

For this example the third-generation epitope sequences were determinedin further 11 environmental allergens (Bosd2, Equc1, Gald4-mutant (withalanine substituted for glycine in position 102), Hevb8, Profillin1-AC,Profillin1-AT, Profillin2-AC, Profillin-birch pollen, Rag weed pollen5and Vesv5), based on their structures sequences (SEQ ID NOS: 12, 13, 15,16, 17, 18, 19, 20, 21 and 22, respectively), their structures(1bj7.pdb, 1ew3.pdb, 1flu.pdb, 1g5u.pdb, 1prq.pdb, 1a0k.pdb, 1f2k.pdb,1cqa.pdb, 1bbg.pdb, and 1qnx.pdb, respectively), and computer modellingof the epitope patterns that had been assembled in our database (shownin Table 8). Further, the epitope sequences of the four environmentalallergens of example 9, Bet v1, Der f2, Der p2, and PhI p2, wereredetermined.

The additional allergens arise from common sources of allergy: cows (Bosd2 which is a bovine member of the lipocalin family of allergens),horses (Equ C1, a major horse allergen also of the lipocalin family),Hen egg white (Lysozyme Gal D 4), Latex (Hey b8, a profilin from Heveabrasiliensis), Acanthamoeba castellani (Profilin1-AC, a profilin isoformIA and Profilin2-AC, a profilin isoform II), Arabidosis thaliana(Profillin1-AT a cytoskeleton profilin), Birch (Profilin-birch pollen(Birch pollen profilin), Rag weed pollen5 (Ragweed pollen allergen Vfrom Ambrosia trifida) and whasp venom (Ves v5 allergen from Vespulavulgaris venom).

The protein surface is scanned for epitope patterns matching the given“consensus” sequence of about 6-12 residues. First, residues on theprotein surface that match the first residue of the consensus sequenceare identified. Within a specified distance from each of these, residueson the protein surface that match the next residue of the consensussequence are identified. This procedure is repeated for the remainingresidues of the consensus sequence. The method is further describedunder the paragraph “Methods” above and the program can be found inAppendixes.

The critical parameters used in this screening included:

-   -   i) a maximal distance between the alpha-carbon atoms of        subsequent amino acids,    -   ii) a minimal accessability of the amino acid of 20 Å2,    -   iii) the largest maximal distance between the most distinct        amino acids should be less than 25 Å,    -   iv) the best epitope were taken,    -   v) the homology with the epitope pattern of interest was 100%

In this way a number of potential epitopes are identified. The epitopesare sorted according to total surface accessible area, and certainentries removed:

-   -   a. Epitopes that contain the same protein surface residue more        than once. These are artefacts generated by the described        algorithm.    -   b. Epitopes which are “too big”, i.e. where a distance between        any two residues in the epitope exceeds a given threshold.

The epitope sequences found were:

Bosd2: Epi#01 L65, P155, P156, R17, R40, N37, Y39, R41, T67 L65, P155,P156, R17, R40, N37, Y39, R41, S52 L64, P155, P156, R17, R40, N37, Y39,R41, T54 Epi#02 T121, K150, S122, R17, P156, Y39, R41, R40 T121, K150,S122, R17, P156, Y56, R36, V30 Epi#03 L128, K130, H92,17, Y76 L134,K130, H92,17, Y76 L128, K130, H92,191, Y76 Epi#04 R72, Y76, S50, Q73,K71, V69, I45 K71, Y76, S50, Q73, R72, V69, L80 K71, Y76, S50, Q73, R72,V69, I42 Epi#06 G14, P13, D47, S10, K11, P9 G14, P13, D47, S10, S94, P9G14, P13, D47, C44, S10, P9 Epi#08 K71, E49, S50, V69, F82 K71, E49,S50, V79, F82 Epi#09 I7, S10, D8, E95, K119, N96, S122, T121 S10, I7,D8, E95, K11, N96, S122, T124 Epi#10 E15, T54, R41, T67, F55, R17, K119E43, T54, R41, T67, F55, R17, K119 E31, T151, N153, C63, F55, R40, R41E31, T151, N153, C154, F55, R41, R17 Epi#11 K26, I145, Q132, E143 K26,I145, Q132, E137 K26, I145, Q132, E129 Epi#12 Y105, E108 Y83, E81 Epi#15N153, P156, D152, I149, T121, G120 R17, P156, D152, I149, T121, G120N153, P156, D152, I149, R17, G14 Epi#18 R109, 1110, G107, Y83, T85, E81,V69 R109, 1110, G107, Y105, T85, E81, V69 Epi#19 E43, N46, S50, Q73,R72, K71 D47, N46, S50, Q73, R72, G75 E49, N46, S50, Q73, R72, K71 I45,N46, S50, Q73, R72, K71 Epi#20 V30, K28, P34, L57, L65, K58, D59, G32,D27 V30, K28, P34, L57, L64, K58, D59, G33, D27 Epi#22 D8, S10, D47,P13, E15 D8, S10, D47, P13, E43 D47, S10, D8, V93, E95 D8, S10, D47,C48, K71 Epi#23 K119, N96, E127, S122, L128, P125 K150, N147, E146, Y20,F123, P125 K11, N96, E127, S122, L128, P125 Epi#24 E129, K130, E126,P125, S122, L128, Q133 E126, K130, E129, P125, S122, R17, K119 E126,K130, E129, P125, T124, L128, Q133 Epi#25 R72, K71, I45, D47, N46 R72,K71, I45, E43, N46 Epi#27 D47, E49, E74, K71 D24, E143, E146, K150 D47,E43, E15, K119 Epi#28 L134, Q133, L128, E126, K130, F123, S122, K150Q132, K130, E126, L128, F123, S122, K150 L65, D59, Q60, K58, E31, L57,G32, D27 G61, D59, Q60, K58, E31, K28, G32, D27 Epi#29 V69, K71, L80,R72, E74 I45, K71, L80, R72, E74 G61, Q60, L64, F55, E68 Epi#30 G120,N96, S94, H92, K130, L128, P125 I91, I7, S94, H92, K130, L128, P125Epi#33 K130, F123, P125, S122, K150 K71, Y76, P9, S10, S94, K119 Epi#34I7, P9, S10, G14, R17, T121, S122 I45, P13, S10, G14, R41, Y39, P156Epi#37 T67, V69, L80, K71, Y76 P156, R40, L65, K58, D59 P155, R40, L65,K58, N153 Epi#38 L80, G84, E108, R109, N25, P141, S136 Epi#39 E137,R138, P141, G139, L134 E31, L57, R36, P34, G84, L80 Epi#40 R17, G120,T121, K150, S122 R17, G120, T121, K150, T151 Epi#41 P34, Y83, L80, V69,S52 P34, Y83, L80, V79, S50 Epi#42 L128, P125, S122, G120, R17, R41L128, P125, S122, G120, R17, R40 Epi#44 S10, D47, P9, Y76, S50, V69, T67I45, D47, P9, Y76, S50, V69, T67 Epi#45 D27, P34, F82, Y105, R109, D106,G107 D59, P34, F82, Y105, R109, D106, G107 K58, P34, F82, Y105, R109,D106, G107 D27, P34, F82, Y105, R109, D106, G84 Epi#46 Y39, R41, R40,P155, C63, Q60 Y20, R17, R40, P155, C63, Q60 Epi#47 L128, E126, E129,L134, R138, Q133, N142, P141, S136 V69, E81, E68, I42, R41, F55, N37,R40, P156 V69, E43, E15, I42, R41, F55, N37, R40, P156 S122, E127, E129,L134, R138, Q133, N142, P141, S136 Epi#48 E43, D47, P13, P9, V93 S10,D47, P9, P13, G14 E43, D47, P13, P9, V90 E49, D47, P13, P9, V93 Equc1:Epi#02 L66, N68, A65, F90, S69, Y72, R64, V89 A65, R64, S31, F28, S112,Y123, R110, V108 L179, R180, Q178, F177, P143, Y38, R141, V145 L66, R64,S31, F28, S112, Y123, R110, V125 L66, N68, A65, F90, S69, Y72, R64, V62Epi#03 K32, A65, I63, Y72 Epi#05 G35, A65, S69, T93, G97, R26, S112,Y123 G35, A65, S69, T93, G97, R26, S112, I25 Epi#07 G97, T93, S70, D91,S100, R110, V125, P132, D128 Epi#08 K129, D130, F127, V108, F90 K129,D130, F127, V108, F109 K129, D130, F127, V125, F136 K129, D130, F127,V125, F133 Epi#10 E48, N53, N80, T77, C83, F177, R175, K172 E82, N80,N53, T77, C83, F177, G181, R180 E52, N53, N80, T77, C83, F177, R175,K172 Epi#11 F133, K47, I167, Q158, V163, E165 Epi#12 Y38, E142 Y38, E36Y139, E142 Epi#13 K129, P132, D45, 1167, Q158, G161 R131, P132, D45,1167, K164, G161 Epi#16 P87, Y72, R64, S70, S69, D67, A65, N68 Epi#17A65, S31, R64, S34 Epi#18 R64, S31, I30, A65, S34, L66, N68, S69 Epi#19E82, N80, C83, Q178, R175, K172 Epi#22 D130, P132, D128, Y106, K129Epi#23 D144, K150, E148, P147, S146, E151, K155 Epi#25 R160, K159, I156,E151, E148 Epi#27 E118, E142, D144, K172 E36, E142, D144, K172 Epi#28I173, D174, Q178, L179, E85, C83, F177, G181, R180 I173, D174, Q178,L179, E85, C83, F177, P143, D144 Epi#29 G181, Q178, L179, R180, E36G181, Q178, L179, R180, E85 Epi#30 I30, N27, S112, H119, I121, I25, V23Epi#31 L122, R110, N27, R26, F28, I30, D96 L124, R110, N27, R26, F28,I30, D96 Epi#33 H119, Y38, V62, S34, S31, R64 Epi#34 V62, P87, M88, V89,R64, S31, S34 Epi#37 P87, V89, L66, R64, D67 Epi#40 R64, L66, A65, Y72,S34 R64, L66, A65, Y72, S69 Epi#41 P132, Y106, L101, V89, S100 P132,Y106, L101, V89, S70 Epi#44 V46, R131, D128, P132, Y106, S100, V89, P87Epi#45 K129, P132, F127, Y106, N102, D91, V89 K129, P132, F127, Y106,N102, D104, G105 Epi#47 S146, E148, E152, V23, R26, A24, N27, R110, S112V23, E115, E118, N116, R26, F28, N27, R110, S112 Gald4: Epi#01 L75, N65,P70, R73, R61, N59, Y53, R45, T47 L75, N65, P70, R68, R61, N59, Y53,R45, T47 Epi#02 A90, N77, L75, R73, P70, R61, R68 A122, R125, Q121,T118, R114, R112 Epi#04 R21, Y20, S24, Q121, R125, R128, L129 R21, Y20,S24, Q121, R125, R128, G126 Epi#05 G16, A10, R128, G126, A122, T118,G117 G4, A10, R128, G126, A122, T118, G117 Epi#06 G67, P79, D66, R61,R73, P70 G67, N65, D66, S72, R73, P70 G49, N46, D48, R61, R73, P70Epi#07 G71, T69, D66, S72, R73, P70, D48 G67, T69, D66, S72, R73, P70,D48 Epi#08 K1, D87, S86, V2, F38 K1, D87, S86, V2, F3 Epi#09 Epi#10 E7,A11, R14, A10, C6, F3, R5, R125 D87, A11, R14, A10, C6, F3, R5, R125T47, N46, N44, S36, F34, R114, R112 D18, A10, R14, A11, C6, F3, R5, R125T118, N113, R112, A110, F34, R114, K116 Epi#11 L129, I124, Q121, V120,D119 Epi#12 Y53, E35 Epi#15 R73, P70, D66, I78, A82 R73, P70, D66, I78,A90 Epi#17 A102, S100, R21, S24 Epi#18 R112, N113, R114, F34, V109,A107, A102, N103 N113, R112, R114, F34, V109, A107, N103, S100 Epi#19D18, N19, S24, Q121, R125, L129 D18, N19, S24, Q121, R125, G126 Epi#22D48, P70, D66, W63, W62 D66, P70, D48, T69, W62 D48, P70, D66, W63, K97Epi#23 R45, N44, E35, N39, Q41, A42 R45, N44, E35, Y53, Q41, A42 Epi#25R128, R125, W123, D119, N27 R128, R125, W123, D119, V120 Epi#26 W62,S72, W63, P79, A82, D87 W62, S72, W63, P79, G67, D66 Epi#28 G117, D119,Q121, I124, E7, C6, F3, A11, R14 A122, D119, Q121, I124, E7, C6, F3,A11, R14 Epi#29 G126, R125, L129, R128, E7 G16, R14, L129, R128, E7Epi#30 I124, L129, A10, H15, I88, L84 I124, L129, A11, H15,188, L84Epi#31 L75, R73, N65, R61, W62, I98, D101 L75, R73, N74, R61, W62, I98,D101 Epi#33 Q41, F38, V2, S86, S85, K1 Q41, F38, V2, S36, A110, R114Epi#34 W63, W62, T69, G71, R73, S72, P70 W62, W63, S72, L75, R73, T69,P70 Epi#36 A110, A107, A102, S100, K96, A90, A82 Epi#37 A10, R128, L129,R14, D18 A10, R128, L129, K13, N19 Epi#40 R128, L129, A11, T89, A90, S85R14, L129, A11, T89, A90, S85 Epi#41 Y53, L84, S81 Y53, L84, S86 Epi#42P79, S81, N65, P70, R61, R73 P79, S81, N65, P70, R61, R68 Epi#44 L129,R14, D18, Y20, S24, V120, T118 L129, R14, D18, Y23, S24, V120, T118Epi#46 L75, R61, R73, P70, N65, G67 L75, R73, R61, P70, N65, A82 L75,R61, R68, P70, N65, G67 Epi#47 S72, G71, R68, N65, R61, L75, N77, R73,P70 G67, S72, R68, N65, R61, L75, N77, R73, P70 Epi#49 D87, L84, Q41,Q57, Y53, T43, N44 D87, L84, Q57, Q41, Y53, T43, N46 D87, L84, Q41, Q57,Y53, T43, N39 Epi#50 R73, W62, W63, P79, S81 R73, W63, W62, S72, P70Epi#51 D18, H15, K13, R14, L129, R125, W123 Epi#52 F34, A110, R112,R114, W111, N27, Q121 F3, A11, E7, R5, W123, D119, Q121 W123, A122,T118, R114, W111, N27, Q121 Hevb8: Epi#01 L20, P109, P112, K86, R84,N116, Y125, Q129, T111 L110, P109, P112, K86, R84, N116, Y125, Q129,T111 Epi#02 A48, K43, Q41, F42, T70, Y72, R84, V74 T21, R19, P109, P112,R84, V74 A49, K43, Q41, F42, T70, Y72, R84, V74 Epi#03 L65, K86, I75,Y72 Epi#05 G30, A48, L60, P62, G58, T63, H66, G69 G58, A61, R84, P112,G113, T111, S89, G88 G80, A81, F54, P79, G58, T63, H66, G69 G77, A81,F54, P79, G58, T63, H66, G69 Epi#06 G58, P79, D55, S59, K52, P57 G80,P79, D55, S59, K52, P57 G77, P79, D55, S59, K52, P57 Epi#07 G17, T5, S2,D16, R19, P109, D107 Epi#08 K52, D45, S44, A49, H66, F42 Epi#10 E78,A81, R96, F54, G58, K52 D55, A81, R96, F54, G80, K52 Epi#11 F54, L60,I83, Q76, V82, E78 Epi#12 Y106, E108 Epi#13 H66, L65, P62, T63, A61,P57, A81, P79, G58 H66, L65, P62, T63, A61, P57, A81, P79, G80 H66, L60,P62, T63, A61, P57, A81, P79, G77 Epi#15 R19, P109, D107, I105, K86, G88Epi#18 R19, G17, P109, S89 Epi#22 D29, S44, D45, A48, K52 D29, M51, D55,P79, E56 D45, M51, D55, P79, E78 D29, S44, D45, A49, K52 D45, M51, D55,P79, E56 D29, M51, D55, P57, E78 D29, M51, D55, P57, E56 D45, M51, D55,P57, K52 D45, M51, D55, P57, E78 Epi#24 D55, K52, E56, P79, F54, E78,Q76 D45, K52, E56, P57, F54, E78, Q76 Epi#25 R84, K86, I105, D107, E108R96, H28, I26, D29, V3 Epi#26 W33, S2, W3, V32, G30, D29 Epi#27 D53,E56, D55, K52 Epi#28 V32, Q41, K43, E46, K52, F54, P57, D55 G69, Q41,K43, E46, K52, F54, P57, D55 Epi#29 G130, Q99, L127, R96, E78 L127, Q99,L131, R96, E78 G98, Q99, L127, R96, E78 Epi#30 G69, L67, A49, H66, K71,L65, P62 G80, M51, A48, H28, Q99, L127, L131 Epi#33 Q41, F42, V32, S31,S44, K43 Q41, F42, V47, S44, A48, K52 Q41, F42, V47, S44, A49, K52Epi#34 I105, P112, S89, L110, R19, T21, S37 I105, P112, T111, L20, R19,T21, S37 Epi#37 T63, A49, L60, K52, D55 P62, V74, L60, K52, D45 P62,A61, L60, K52, D55 Epi#38 G77, E78, R96, V82, R84, N116, P112, S89Epi#39 A48, E46, H66, T63, P62, G58, L60 A49, E46, H66, T63, P62, G58,L60 Epi#40 R19, L110, G113, T111, P109, S89 R19, L110, G113, T111, P112,S89 Epi#41 P62, L65, V47, S44 P109, Y106, L110, S89 P112, Y106, L110, S8Epi#44 L20, R19, D16, W3, Y6, S2, G17, P109 L110, R19, D16, W3, Y6, S2,G17, P109 Epi#45 K52, P57, F54, R96, D124, L127 D55, P79, F54, R96,D124, L131 Epi#47 I75, G77, E78, V82, R84, N116, P112, S89 I75, G77,E78, I83, R84, N116, P112, P109 Epi#48 E78, Q76, P79, P57, G58 E78, Q76,P79, P57, G80 E78, Q76, P79, P57, G77 Epi#50 D9, W3, W33, S2, T5 D16,W3, W33, S2, T5 Epi#51 R19, H18, E108, S89, K87, K71, H66 R19, H18,E108, D107, K87, K71, H66 Profillin1-AC: Epi#01 L116, N111, P106, K80,K81, N101, S83, Q105, T108 L116, N111, P106, K80, K81, N101, Y100, Q105,S83 Epi#02 T44, N51, P54, R56, T69, Y78, R71, V68 L24, K93, S92, R75,S76, Y78, R71, R56 Epi#03 L24, K93, I121, Y119 L24, K90, I121, Y119Epi#04 K80, Y100, S83, Q105, K103, N101, G82 K80, Y100, S83, Q105, K103,T17, G12 K80, Y100, S83, Q105, K103, T17, G14 Epi#05 G34, A33, A36, T38,G64, A63, H66, V68 G34, A33, S32, T17, G12, T4, S1, Y5 Epi#06 A46, N50,D53, R56, A57, P54 A52, N50, D53, R56, A57, P54 A72, N50, D53, R56, A57,P54 A57, P54, D53, S47, Q43, P39 Epi#07 G64, T38, D61, S58, R56, A57,P54, D53 G64, T38, D61, S58, R56, A52, P54, D53 Epi#08 K103, E102, G82,V68, H66, F60 K81, E102, G82, V68, H66, F60 Epi#09 L24, S47, D53, A57,V68, R71, L70, R56, N51, N50, R75 L24, S47, D53, A57, V68, R71, L70,R56, N51, T44, T38 Epi#10 D74, N50, N51, T44, F60, R56, R71 D53, N50,N51, T44, F60, R56, R71 Epi#11 F125, K93, I121, Q123, D118 F125, K90,I121, Q123, D118 F49, K90, I121, Q123, D118 Epi#12 Y119, E114 Y100, E102Epi#13 A57, R56, P54, T44, A40, P39, A36, G64, Y67 S58, A57, P54, T44,A40, P39, A36, G64, Y67 Epi#15 N51, P54, D53, I55, R56, A57 R56, P54,D53, I55, T69, A57 R56, P54, D53, I55, T44, A40 Epi#16 Q105, P106, Y100,G14, Q18, S32, A36, A33, D7 Q105, P106, Y100, G14, Q18, S32, A36, A63,D61 Epi#17 A110, S76, R75, S92 A72, S76, R75, S92 Epi#18 N51, N50, R75,S92, L24, S47, T44, P39, N27 N51, N50, R75, S92, L24, T28, T38, P39, N27Epi#22 D53, S47, D25, L24, K93 D53, S58, D61, V68, K81 Epi#23 K103,N101, E102, S83, Q105, P106 K103, N101, E102, S83, Q105, A84 Epi#24E114, K115, A110, P106, S83, E102, K103 D53, G59, A57, P54, R56, L70,K80 E102, K103, A15, P106, S83, A84, Q105 Epi#25 R71, R56, 155, D53, N50R71, R56, 155, D53, N51 Epi#28 I104, Q105, K103, E102, K81, S83, K80G107, Q105, K103, E102, K81, G82, K80 A84, Q105, K103, E102, K81, S83,K80 A110, Q105, K103, E102, K81, S83, K80 Epi#29 I121, K115, L116, E114V112, K115, L116, E114 Epi#30 G59, I55, S58, H66, K80, L70, V68 G59,I55, S58, H66, K80, P106, V99 Epi#33 K80, Y78, V68, S58, A57, R56 K81,Y67, V68, S58, A57, R56 Epi#34 I55, P54, S58, V68, R71, Y78, P106 W29,W2, T4, V11, G12, Y5, S1 Epi#36 A63, A36, A33, V11, G14, Y100, S83,Q105, K103, P106, A110, A15 A63, A36, A33, V11, G14, Y100, T108, Q105,K103, P106, A15, A110 Epi#37 A57, R56, L70, R71, Y78 A57, V68, L70, R56,D53 Y78, R71, L70, R56, N51 P54, R56, L70, R71, D73 T69, R71, L70, R56,D53 Epi#38 G82, E102, A84, V99, N101, P106, S83 Epi#40 R71, L70, A72,Y78, K80, S83 R71, L70, G59, T69, K81, S83 R56, L70, A72, T69, K81, S83Epi#41 P106, Y78, L70, V68, S58 Epi#42 P54, S47, N51, R56, R71 P54, S58,G59, R56, R71 Epi#44 S83, Q105, P106, Y78, A110, G107, T108 V68, R71,D73, Y78, A110, G107, T108 L70, R71, D73, Y78, A110, V112, T108 L70,R71, D73, Y78, A110, G107, P106 Epi#45 K81, H66, F60, R56, D53, G59 K80,H66, F60, R56, D53, G59 D61, H66, F60, R56, D53, G59 Epi#46 L70, R71,R56, P54, N51, A52 L70, R71, R56, P54, N51, A72 V68, R71, R56, P54, N51,A46 Y78, R71, R56, P54, G59, A57 Epi#47 V68, A57, R56, L70, R71, A52,N51, P54, S58 S58, A57, R56, L70, R71, A72, N51, P54, S47 Epi#49 D25,L24, Q43, Q41, T44, N51 D25, L24, Q43, Q41, T38, N27 Epi#50 D7, W2, W29,S1, T4 D7, Y5, W2, W29, S1 Epi#51 K80, H66, D61, T44, P39, T28, W29 K80,H66, D61, T38, P39, T28, W29 Profillin 1-AT: Epi#01 P109, P89, K86, R84,N116, Y106, Q114, T111 Epi#02 L42, K43, Q45, F66, T63, Y72, R84, V74L42, K43, Q45, F66, T63, Y72, R84, V82 Epi#03 K96, I127, Y125 K86, I75,Y72 Epi#05 G77, A81, F54, P57, G58, A61, T63, V74 G58, A61, F59, P57,G77, A81, T97, G80 G80, A81, F54, P57, G58, A61, T63, Y72 Epi#06 G17,P109, D107, T21, K38, P40 G112, P109, D107, T21, K38, P40 G88, P89,D107, T21, K38, P40 Epi#08 K52, E55, G58, V74, F66 K51, E55, G58, A61,F59 Epi#09 D29, D48, K52, F59, A61, T63 D29, D48, K51, F59, A61, T63Epi#10 E108, T111, N18, T21, F39, G68, K71 E108, T111, N18, T21, F105,G112, K86 Epi#11 F105, K86, I75, Q76, V82, E78 F66, K43, I47, Q28, V32,D29 F59, K52, I47, Q28, V32, D29 Epi#12 Y125, E130 Y125, E128 Epi#15K43, P44, D29, I47, K52, G58 K43, P44, D48, I47, Q45, G49 K43, P44, D29,I47, K51, G80 Epi#20 K38, P40, F39, L42, K43, D48, G30, D29 K51, P57,F59, L60, K52, D48, G30, D29 Epi#22 D48, P44, D29, V32, W33 D48, P44,D29, V32, W3 Epi#24 D29, K51, E56, P57, F59, E55, Q79 D48, K52, E55,P57, F59, E56, Q79 Epi#25 R121, K95, I83, D53, E55 R121, K95, I83, E78,V82 Epi#26 W33, S2, W3, V32, G30, D29 Epi#27 E128, E130, D124, K96 E130,E128, D124, K95 Epi#28 I75, Q76, E78, Q79, P57, K51 A61, Q76, E78, Q79,P57, K52 V32, D29, Q99, E130, I127, S129, D124 V32, D29, Q99, 1127,E128, S129, D124 Epi#29 V32, Q41, L42, F66, E70 G69, Q41, L42, F66, E70G68, Q41, L42, F66, E70 Epi#30 G17, N18, H19, Q114, L117, V15 G17, M110,H19, Q114, L117, V15 G113, M110, H19, Q114, L117, V15 Epi#33 Q41, F39,P40, S36, A37, K38 Epi#34 V74, P62, M73, G88, P89, Y106, T111 Epi#37T111, V15, L117, R121, Y125 T111, V15, L117, R121, D124 Epi#39 A81, E55,P57, G58, L60 A81, E78, P57, G58, L60 Epi#40 R121, L117, G112, Y106,P109, T111 R121, L117, G112, Y106, P89, T111 Epi#41 Y125, L131, S129Epi#44 I75, R84, Y72, A61, G58, P62 I75, R84, Y72, A61, V74, T63 Epi#45K38, P40, F105, Y106, N18, D14, G17 K38, P40, F105, Y106, N18, D107, G88K38, P40, F105, Y106, N18, D14, V15 Epi#48 E16, H19, P109, P89, G88 E16,H19, P109, P89, G112 Epi#49 D124, L131, Q99, Q28, T97, N98 D124, L131,Q99, Q28, T97, K96 Epi#50 D9, Y6, W3, W33, S2 D9, W3, W33, S2, S5 D9,W3, W33, V32, S31 Epi#51 D14, H19, E108, T111, L117, R121, H10 D107,H19, E16, Q114, L117, R121, H10 D14, H19, D107, T21, K38, Q35, W33Profillin2-AC: Epi#01 L116, N111, P106, K80, K81, N101, S83, Q105, T108L116, N111, P106, K80, K81, N101, S83, Q105, T108 Epi#02 T53, N58, S57,R56, T69, Y67, R66, V68 T53, K50, A52, R56, T69, Y67, R66, V68 T53, K50,A72, R56, T69, Y67, R66, V68 Epi#03 L116, K115, I121, Y119 Epi#04 K81,Y100, S83, Q105, K103, T17, G12 K80, Y100, S83, Q105, K103, A84, G82K81, Y100, S83, Q105, K103, T17, G14 K80, Y100, S83, Q105, K103, N101,I104 K81, Y100, S83, Q105, K103, A15, G107 Epi#06 A54, N47, D25, T28,A36, P39 A40, N27, D25, T28, A36, P39 A44, N47, D25, T28, A36, P39 G34,A33, D7, T31, A36, P39 A43, N47, D25, T28, A36, P39 Epi#08 K103, E102,G82, V68, F60 K103, E102, G82, V68, F60 K81, E102, G82, V68, F60 Epi#10T53, N58, R56, S57, F60, R66, K81 E61, N58, R56, S57, F60, R66, K80Epi#11 F125, K93, I121, Q105, E102 F125, K93, I121, Q123, D118 Epi#12Y100, E102 Y119, E114 Epi#13 A52, A44, P39, A43, H24, S92, G124, Y119A46, A44, P39, A43, H24, S92, G124, Y119 Epi#15 K103, P106, D118, I121,K93, G124 K103, P106, D118, I121, Q105, G107 K103, P106, D118, I121,Q123, G122 Epi#16 Q105, P106, Y78, R71, S57, N58, A54, A44, D51 Q105,P106, Y78, R71, R56, D51, D74, A52, N47 Epi#18 R66, N58, R56, S57, V68,G82, S83, E102, N101 R66, N58, R56, S57, V68, G82, S83, P106, N101Epi#22 D74, A52, D51, T53, K50 D25, A44, D51, T53, K50 D74, A46, D51,T53, K50 D74, A72, D51, T53, K50 Epi#23 K103, N101, E102, S83, Q105,P106 K103, N101, E102, S83, Q105, A84 Epi#24 D74, K81, A84, P106, S83,E102, K103 D74, K81, E102, P106, T108, A15, K103 Epi#25 R66, K81, E102,N101 Epi#28 I121, D118, Q105, K103, E102, K81, G82, D74 G107, D118,Q105, K103, E102, K81, G82, D74 G122, D118, Q105, K103, E102, K81, G82,D74 Epi#29 I121, K115, L116, E114 V112, K115, L116, E114 Epi#30 I55,N47, A44, H24, K93, I121, L116 I55, N47, A43, H24, K93, I121, L116Epi#31 R56, N58, R66, F60, V68, I55, D51 R66, N58, R56, F60, V68, I55,D51 Epi#33 K115, Y119, P106, S83, A84, K103 Q123, Y119, P106, S83, A84,K103 K81, Y67, V68, S57, A54, R56 K80, Y78, V68, S57, A54, R56 Epi#34W29, W2, T8, V11, G12, T4, S1 W29, W2, T4, G12, G14, T13, T8 Epi#37T108, V112, L116, K115, Y119 T108, A110, L116, K115, N111 T13, V112,L116, K115, D118 P106, A110, L116, K115, N111 Epi#38 G64, E61, A40, V37,N27, P39, S38 G82, E102, A84, V99, N101, P106, S83 Epi#39 A110, E114,T108, P106, G122, L116 Epi#40 G14, G12, T17, K103, S83 R56, A52, T53,A54, S57 R66, A63, T65, K81, S83 R56, A72, T53, A54, S57 R56, G59, T53,A54, S57 R66, G64, Y67, K81, S83 Epi#42 P106, S83, G82, R75, R71 Epi#44S1, Q3, D7, W2, Y5, S32, G12, T8 S1, Q3, D7, W2, Y5, A30, A36, P39 S1,Q3, D7, W2, Y5, S32, V11, T8 S1, Q3, D7, W2, Y5, S32, G12, T4 S1, Q3,D7, W2, Y5, A30, A33, T31 S1, Q3, D7, W2, Y5, A30, A36, T28 S1, Q3, D7,W2, Y5, S32, G12, T13 S1, Q3, D7, W2, Y5, S32, G34, T31 Epi#45 K93, H24,F49, R75, D74, G82 D25, H24, F49, R75, D74, G82 Epi#47 A36, G64, E61,A40, A44, A54, N58, R56, S57 Epi#50 D7, Y5, W2, T8, S1 D7, W2, W29, T28,P39 Epi#51 K90, H24, K93, D25, P39, T28, W29 T91, H24, K93, D25, P39,T28, W29 Profillin-Brich Pollen: Epi#01 L124, N118, P114, K88, K73, H68,Y74, R86, T95 Epi#02 T113, N118, Q116, P114, R86, V76 T50, K54, L62,T65, Y74, R86, V84 Epi#03 L133, K98, I129, Y127 Epi#04 S40, Q43, K45,T50, G32 S40, Q43, K45, T50, G51 S40, Q43, K45, T50, I49 Epi#05 G82,A81, A83, P59, G60, A63, T65, V76 G82, A83, A81, P59, G60, A63, H61, V76G79, A81, A83, P59, G60, A63, T65, V76 Epi#06 G70, P46, D31, T50, K54,P59 A81, P59, D55, T50, Q47, P46 G32, P46, D31, T50, K45, P42 G51, P46,D31, T50, K54, P59 Epi#08 A81, E57, G60, A63, H61, F56 A81, E57, G60,V76, H68, F44 K54, E57, G60, A63, H61, F56 Epi#11 F56, K98, I85, Q78,V84, E122 F56, K98, I27, Q37, V34, D31 F56, K97, I85, Q78, V84, E80Epi#12 Y6, E9 Y127, E122 Epi#13 H68, L62, P64, T65, A63, P59, A81, G82,G79 H61, L62, P64, T65, A63, P59, A81, G79, F56 H68, L62, P64, T65, A63,P59, A83, G79, G60 Epi#15 K45, P46, D31, I49, Q47, G32 K45, P46, D31,I49, K54, G60 K45, P46, D31, I49, K54, G82 K45, P46, D31, I49, T50, G51Epi#16 Q116, P114, Y108, M12, S39, S40, A23, A24, D8 Q116, P114, Y108,M12, Q37, S40, A23, A24, D8 R86, P114, Y108, M12, S39, S40, A23, A24, D8Epi#22 D126, L133, D130, Y127, E122 D130, L124, D126, Y127, E122 D130,L128, D126, Y127, E122 Epi#23 R123, N118, E122, L124, L11, A23 R123,N118, E122, L124, L11, A36 R123, N118, E122, L124, L11, A24 Epi#24 E109,G90, E110, P114, R86, E80, Q78 E57, K54, E58, P59, F56, A81, Q78 E58,G60, E57, P59, F56, E80, Q78 Epi#25 R86, K88, I107, E109, E110 R86, K88,I77, E80, V84 R86, K88, I107, E109, V112 Epi#27 57, E58, D55, K54 D55,E57, E58, K54 Epi#28 V34, D31, Q101, K98, E122, L128, Q131, G132, D130I129, D126, Q131, L128, E122, K98, Q101, G100, D130 I72, H68, Q47, F44,E48, K45, Q43, G70, K73 I72, H68, Q47, I49, E48, K45, Q43, G71, K73Epi#29 I129, Q101, L128, R123, E122 G132, Q131, L128, R123, E122 Epi#30I77, M75, A63, H61, P59, L62, P64 G90, M75, A63, H61, K54, L62, P64Epi#33 Q116, Y108, P111, S91, K89 K88, Y108, P111, S91, K8 Epi#34 V76,P64, M75, L62, G51, T50, P46 I27, W35, S33, V34, G32, T50, P46 V76, P64,T65, L62, G51, T50, P46 Epi#35 A24, L22, A23, S39, M12, 1107 A23, L11,A36, S39, M12, I10 Epi#37 Y127, R123, L124, K97, N118 Y108, A23, L11,R123, Y127 A23, A24, L11, R123, Y127 Epi#39 A81, E57, H61, T65, P64,G60, L62 A81, E58, H61, T65, P64, G60, L62 Epi#40 R123, L11, A23, Y108,P111, S91 R123, L11, A24, Y108, P111, T113 Epi#41 P111, Y108, L22, V112,S91 P114, Y108, L22, V112, S91 Epi#43 I27, W35, A36, L11, Q37, S39, M12,1107, T95 Epi#44 I77, R86, P114, Y108, S91, V112, P111 V120, Q116, P114,Y108, S91, V112, P111 L22, Q116, P114, Y108, S91, V112, T113 L22, Q116,P114, Y108, A23, V112, P111 Epi#47 I129, Y127, E122, M119, R123, L124,N118, R86, P114 L133, Y127, E122, M119, R123, L124, N118, R86, P114Epi#48 E122, Q116, P114, P111, V112 S91, K88, P114, P111, V112 Epi#50H10, Y6, W3, S2, T5 H10, Y6, W3, T5, S39 Epi#51 K73, H68, K45, Q47, P46,S33, W35 Q101, H30, D31, T50, K45, Q47, H68 Rag Weed Pollen5: Epi#03 L4,K37, A33, I34, Y17 L4, K37, A33, I34, Y29 Epi#05 A33, N36, T40, G3, S20,L4 A33, N38, T40, G3, S20, Y25 A33, N36, G3, T40, S20, I22 Epi#06 A33,N36, D2, C19, K24, P21 A33, N38, D2, S20, K24, P21 Epi#09 I22, L4, D2,N38, D1, K37, A33, N36, T40 T9, G15, E7, V14, D30, K32, N36, T40, L4 T9,G15, E7, V14, D30, K32, N38, N36, L4 Epi#12 Y17, E7 Y6, E7 Epi#20 V27,K24, P21, L4, K37, D2, G3, D1 V27, K24, P21, L4, N36, D2, G3, D1 Epi#22D1, D2, L4, K37 D1, D2, P21, K24 D2, L4, T40, D1 Epi#23 N10, E7, Y6, L4,P21 Epi#25 K32, I34, D30, V14 K37, I34, D30, V14 K16, I34, D30, V14Epi#33 K32, Y17, V27, S20, K24 K16, Y6, P21, S20, K24 Epi#34 I22, P21,S20, V27, G12, Y17, T9 I22, P21, S20, V27, G12, Y29, S31 Epi#40 G12,G15, Y29, K37, T40 G15, G12, Y17, K16, T9 G12, G15, Y29, K32, S31 Epi#41P21, Y6, L4, S20 Epi#44 L4, D2, P21, Y25, S20, V27, T40 L4, D2, P21,Y25, S20, G3, T40 Vesv5: Epi#01 L59, P67, P65, K143, K144, N64, Y140,R62, T61 L59, P67, P70, R57, K204, N73, Y201, Q202, T203 L59, P67, P69,R57, K72, N73, Y201, Q202, T203 L152, N149, P142, K145, K143, N64, Y140,R62, T61 Epi#02 L9, K7, Q108, P191, Y107, R102, V13 L9, K7, Q108, S192,Y107, R102, V13 Epi#03 L9, K7, A105, I6, Y3 Epi#04 K106, Y107, S192,Q108, K7, A105, I6 K106, Y107, S192, Q108, K7, V13, G12 Epi#05 G58, A56,R57, P69, G66, R62, T61, L59 G58, A56, R57, P69, G63, R62, T61, L59Epi#06 G66, N64, D139, R62, K138, P67 G66, N64, D139, R62, K138, P65G63, N64, D139, R62, K138, P67 Epi#08 K145, E199, S147, F151 K196, E198,S147, F151 K144, E199, S147, F151 Epi#09 L152, D150, S147, K144, N64,T61, L59 L152, D150, D139, K153, F151, S147, N197 D139, N64, R62, D135,K153, F151, S147, N197 Epi#10 E199, N197, N194, S147, F151, G148, K143E199, N197, N194, S147, F151, G148, K196 E199, N197, N194, S147, F151,G148, K145 Epi#11 K179, I176, Q177, V30, E178 K29, I176, Q177, V30, E178Epi#12 Y201, E199 Epi#13 S147, L200, P142, T203, A56, P70, L59, P67, G66S147, L200, P142, T203, A56, P69, L59, P67, G58 S147, L200, P142, T203,A56, P70, L59, P67, G63 S147, L200, P142, T203, A56, P69, L59, P67, Y140Epi#15 K106, P191, D103, I6, K5, A105 K106, P191, D103, I6, K7, G12Epi#16 R57, P70, Y201, M74, Q53, N76, D50, A56, N73 R57, P69, Y201, M74,Q53, N76, D50, A56, N73 Q108, P191, Y107, R102, Q111, S192, D103, A105,N2 Epi#18 R57, L59, T61, P67, N64 R57, L59, T61, P65, N64 Epi#19 E167,N164, S192, Q108, R102, K7 E198, N194, S192, Q108, R102, K7 D103, T100,C8, Q108, R102, K7 Epi#22 L9, D103, T100, K10 A105, D103, L9, K7 D50,L45, D43, T37, K38 S147, D150, L152, K153 Epi#23 K196, N197, E199, N164,Q202, P70 K145, N197, E199, N164, Q202, P69 Epi#24 E198, K196, E199,P142, T203, P69, K143 E198, K145, E199, P142, T203, P70, K204 E198,K196, E199, P142, T203, P70, K72 E198, K145, E199, P142, F146, F151,K196 Epi#25 R57, K54, D50, N76 R57, K54, D50, E47 Epi#27 D43, E40, D125,K122 D50, E47, D43, K38 Epi#28 Q202, E199, K196, F151, S147, K144 Q202,E199, K196, F195, S147, K145 Epi#29 G58, R57, L59, R62, E136 G148, K145,L200, F195, E199 G148, K145, L200, F195, E198 Epi#33 K23, Y19, P24, S21,A16, K18 K23, Y34, P24, S21, A16, R102 Epi#34 I176, W180, T116, L115,G117, T119, S118 V31, P24, S21, L22, G35, Y34, T37 Epi#37 P69, R57, L59,K54, D50 P70, R57, L59, R62, D135 A56, R57, L59, R62, N64 P69, R57, L59,R62, D139 Epi#39 E199, L200, T203, P70, G58, L59 E198, L200, T203, P69,G58, L59 Epi#40 R57, L59, G58, T203, P69, T61 R57, L59, A56, Y201, K204,T203 R57, L59, A56, Y201, K72, T203 Epi#41 P24, Y19, L22, S21 P24, Y34,L36, S33 Epi#42 P191, S192, Q111, H98, R102, Q108 Epi#44 L59, R57, P70,Y201, A56, G58, T61 L59, R57, P69, Y201, A56, G58, T203 L59, R57, P70,Y201, A56, G58, P67 Epi#45 K153, H156, F151, Y140, N149, D150, L152D135, H156, F151, Y140, N141, D150, L152 K143, P142, F146, Y140, N149,D150, L152 Epi#47 G58, L59, R57, M74, A56, Q202, N73, P70, P69 G148,Y140, R62, L59, R57, A56, N73, P70, P67 G66, G63, R62, L59, R57, A56,N73, P70, P67 G155, E136, R62, L59, R57, A56, N73, P70, P67 Epi#48 Q202,K204, P69, P67, G58 Q202, K204, P70, P67, G63 Q202, K72, P70, P67, G66Epi#49 D125, D43, L45, V78, Q42, Q39, T37, K38 D125, D43, L45, V78, Q42,Q39, T37, K41 Epi#50 H98, Y96, W90, L22, S21 H98, Y96, W90, P24, S33Epi#52 F0, A16, R102, W90, N25, Q95 F0, A16, R102, W90, N25, Q93 Betv1:Epi#03 SAS: 270, Size 11.07: L24, K20, H76, I23, Y81 SAS: 204, Size11.96: L24, K20, A16, I23, Y81 Epi#05 SAS: 298, Size 14.01: G110, A106,A16, P14, G111, T10 SAS: 242, Size 14.01: G110, A106, A16, P14, G111,T107 Epi#08 SAS: 464, Size 11.12: K123, E127, G1, H121, F3 SAS: 455,Size 12.95: K129, E127, G1, H121, F3 SAS: 438, Size 13.31: K123, D125,G1, H121, F3 SAS: 428, Size 11.12: K123, E127, V2, H121, F3 SAS: 425,Size 11.65: K123, E127, G124, H121, F3 Epi#09 SAS: 466, Size 20.55:D109, A106, V105, K80, A16, T77 SAS: 444, Size 20.55: D109, G110, V105,K80, A16, T77 SAS: 427, Size 20.55: D109, G111, V105, K80, A16, T77 SAS:398, Size 19.17: T10, G110, V105, K80, A16, T77 SAS: 381, Size 19.17:T10, G111, V105, K80, A16, T77 Epi#10 SAS: 558, Size 15.18: D75, T77,N78, A106, F79, R17, K20 SAS: 549, Size 21.96: E6, T7, N4, F3, G1, K123SAS: 517, Size 13.31: D75, T77, N78, A16, F79, R17, K20 SAS: 497, Size15.13: D75, T77, N78, A16, F22, R17, K20 Epi#12 SAS: 335, Size 9.08: T7,Y5, E6, N4 SAS: 331, Size 11.28: R145, Y150, E148, L152 SAS: 326, Size10.37: R70, Y83, E73, P50 SAS: 311, Size 10.32: 1116, Y5, E6, N4 SAS:308, Size 8.33: R145, Y150, E148, S149 Epi#18 SAS: 328, Size 24.67:S117, K103, F79, V105, A16, Y158, L24 Epi#22 SAS: 533, Size 9.96: D125,D93, K123, E127 SAS: 533, Size 9.96: D93, D125, K123, E127 SAS: 476,Size 11.40: D125, D93, K123, E96 SAS: 476, Size 11.40: D93, D125, K123,E96 SAS: 400, Size 17.99: D125, D93, P90, E87 Epi#23 SAS: 451, Size22.02: K68, N43, E42, S57, F64, P63 SAS: 450, Size 22.02: K55, N43, E42,S57, F64, P63 SAS: 428, Size 22.02: K68, N43, E42, S57, L62, P63 SAS:427, Size 22.02: K55, N43, E42, S57, L62, P63 SAS: 412, Size 18.85: K68,N43, E42, S40, F30, P35 Epi#24 SAS: 734, Size 18.92: E127, K123, E96,P90, S136, E131, K129 SAS: 729, Size 18.92: D93, K123, E96, P90, S136,E131, K129 SAS: 716, Size 19.57: E127, K123, E96, P90, S136, E131, K134SAS: 711, Size 19.57: D93, K123, E96, P90, S136, E131, K134 SAS: 708,Size 20.49: D125, K123, E96, P90, S136, E131, K129 Epi#25 SAS: 467, Size12.68: R70, K55, I44, E42, E45 SAS: 425, Size 12.68: R70, K54, I44, E42,E45 SAS: 420, Size 14.01: R70, K55, I44, D27, E42 Epi#27 SAS: 613, Size14.25: D93, E127, A130, E131, K129 SAS: 595, Size 16.54: D93, E127,A130, E131, K134 SAS: 592, Size 16.70: D125, E127, A130, E131, K129 SAS:574, Size 19.79: D125, E127, A130, E131, K134 SAS: 524, Size 18.78: D93,E127, A130, E131, K137 Epi#28 SAS: 869, Size 21.93: V33, Q36, F58, E60,L62, F64, P63, K65 SAS: 837, Size 21.83: V33, Q36, F58, E60, L62, F64,G61, K65 SAS: 808, Size 24.56: V33, Q36, F58, E60, L62, F64, P90, K65SAS: 783, Size 21.83: V33, Q36, F58, E60, K65, F64, S57, K68 SAS: 782,Size 21.83: V33, Q36, F58, E60, L62, F64, S57, K65 Epi#29 SAS: 516, Size9.52: G61, K65, L62, E60 SAS: 440, Size 8.70: G61, P63, L62, E60 SAS:371, Size 6.78: G61, P59, L62, E60 Epi#32 SAS: 374, Size 17.88: F79,A16, A106, D109, V12 SAS: 354, Size 20.42: F22, A16, A106, D109, V12Epi#33 SAS: 541, Size 18.79: K65, F64, P90, S136, A135, K134 SAS: 498,Size 9.15: Q36, F30, P35, S39, K32 SAS: 496, Size 11.27: Q36, F30, P35,S40, K32 SAS: 494, Size 12.19: Q36, F58, P35, S39, K32 SAS: 493, Size18.79: K65, Y66, P90, S136, A135, K134 Epi#36 SAS: 447, Size 19.17: T77,A16, A106, V12, G110, T10 SAS: 430, Size 19.17: T77, A16, A106, V12,G111, T10 SAS: 392, Size 19.17: T77, A16, A106, V105, G110, T10 SAS:391, Size 19.17: T77, A16, A106, V12, G110, T107 SAS: 375, Size 19.17:T77, A16, A106, V105, G111, T10 Epi#40 SAS: 246, Size 21.55: A106, A16,Y158, S155 SAS: 223, Size 13.25: A135, A130, Y5, T7 SAS: 196, Size14.88: A135, A130, Y5, S117 SAS: 178, Size 10.62: A135, G140, T142, S136Epi#44 SAS: 530, Size 19.04: L24, R17, D156, Y150, S149, V12, T10 SAS:492, Size 19.04: 123, R17, D156, Y150, S149, V12, T10 SAS: 490, Size17.39: L24, R17, D156, Y150, S149, V12, P14 SAS: 483, Size 23.09: L24,R17, D156, Y158, A16, A106, P108 SAS: 474, Size 20.83: L24, R17, D156,Y150, S149, V12, T107 Epi#45 SAS: 606, Size 21.41: K32, P35, F30, Y150,R145, V12 SAS: 546, Size 20.89: K32, P31, F30, Y150, R145, V12 SAS: 533,Size 15.19: K32, P35, F30, Y150, R145, G140 SAS: 533, Size 12.63: K32,P35, F30, Y150, R145, V33 SAS: 532, Size 19.60: K32, P35, F30, N28, D27,I44 Epi#47 SAS: 333, Size 21.03: R17, L24, N28, P31, P35 SAS: 300, Size22.72: R17, L24, N28, P31, S39 SAS: 298, Size 21.80: R17, L24, N28, P31,S40 SAS: 269, Size 24.87: R17, L24, N28, P31, S57 Epi#48 SAS: 436, Size14.26: S57, K65, P90, P63, G61 SAS: 414, Size 17.96: S39, K32, P35, P59,G61 SAS: 412, Size 17.96: S40, K32, P35, P59, G61 SAS: 389, Size 18.32:S57, K65, P63, P90, G92 SAS: 365, Size 21.15: S57, K65, P59, P35, V33

“SAS” is solvent accessible surface. “Size” is the total surface area ofthe epitope in A2.

Derf2: Epi#02 A98, K100, S101, P99, R128, R31 A98, K100, R128, P99, R31,V94 T91, N93, P95, P34, R31, R128 L61, N93, P95, P34, R31, R128 Epi#03L40, K15, A39, I13, Y86 L40, K14, A39, I88, Y90 Epi#05 G32, A98, R31,P34, G20, T36, T91, Y90 G32, A98, R31, P34, G20, T36, T91, V94 G32, A98,R31, P34, G20, T36, T91, L37 G32, A98, R31, P34, G20, T36, T91, V18Epi#06 A98, P99, D129, R31, K96, P95 G32, P99, D129, R128, R31, P95 A98,P99, D129, R31, K33, P95 A98, P99, D129, R31, K96, P34 A98, P99, D129,R128, K126, P26 Epi#07 T107, S57, D59, S101, R128, A98, P99, D129 T107,S57, D59, S101, R31, A98, P99, D129 Epi#08 K15, D87, V76, H74, F75 K14,D87, V76, H74, F75 K77, D87, V76, H74, F75 Epi#09 L61, D64, I68, H74,F75, T70, N71 N114, N46, D113, K48, N71, T70, T49 G83, N46, D113, K48,N71, T70, T49 Epi#10 L40, I13, D42, N44, V81, K48, N46, N114, G115 L40,I13, D42, N44, V81, K82, N46, N114, G115 L37, D19, G20, V18, V3, D4, K6,A120, T107, V105 Epi#11 F75, K51, I111, Q45, V116, D113 F75, K51, I111,Q45, V81, D113 Epi#12 Y90, E38 Epi#13 H30, R31, P95, A98, P99, S101,G60, L61 Epi#15 K96, P99, D129, 128, R128, A98 K96, P99, D129, I127,R128, A98

K96, P99, D129, 129, R128, A98

K55, P66, D64, I68, T70, G67 Epi#18 R31, R128, I28, G125, T123, H124,V105 R31, R128, I127, G125, T123, H124, V105 Epi#22 D1, M17, D4, V3, K6D1, M17, D19, P34, K96 D1, M17, D4, V5, K6 Epi#23 K14, N11, E12, N44,Q85, P79 K14, N11, E12, N10, Q45, P79 K14, N11, E12, N44, Q84, P79 K14,N11, E12, L40, Q85, P79 Epi#24 D129, K100, E102, P99, R128, R31, K96E62, G60, E102, P99, R128, R31, K96 D129, K126, E102, P99, R128, R31,K33 D129, K126, E102, P99, R31, P95, K96 Epi#25 R31, K96, I97, D59, E62R128, R31, 197, D59, E102 R128, K126, I127, E102, N103 Epi#27 D64, E62,D59, K100 D59, E62, D64, K55 D87, E38, D19, K33 D19, E38, D87, K15 D19,E38, D87, K14 D19, E38, D87, K77 Epi#28 V16, D87, Q85, K14, E12, K15,Q2, D1 I13, D87, Q85, K14, E12, K15, Q2, D1 V3, D1, Q2, K15, E12, K14,Q85, D87 L40, D87, Q85, K14, E12, K15, Q2, D1 I88, D87, Q85, K14, E12,K15, Q2, D1 V76, D87, Q85, K14, E12, K15, Q2,D1 V18, D1, Q2, K15, E12,K14, Q85, D87 Epi#29 G32, N93, L61, E62 V94, N93, L61, E62 Epi#30 G60,I97, A98, H30, K96, P34, P95 I68, N71, H74, K77, P79, V81 G32, I97, A98,H30, K96, P95, P34 Epi#34 V105, P26, S24, G125, R128, S101, P99 W92,P34, T91, V94, R31, S101, P99 I28, P26, T123, G125, R128, S101, P99Epi#37 A120, V16, L40, K14, N11 A39, V16, L40, K14, N11 Y90, A39, L40,K14, N11 Y86, A39, L40, K14, N11 Epi#39 A120, E38, T91, P34, G20, L37A39, E38, T91, P34, G20, L37 Epi#40 G20, L37, A120, T123, K6, S24 A39,L37, A120, T123, K6, S24 G20, L37, A120, T107, K6, T123 Epi#41 P34, L37,V106, S57 Epi#42 P26, S24, G125, R128, R31 P99, S101, G125, R128, R31Epi#44 V16, Q2, D19, P34, W92, Y90, A39, V18, T91 V16, Q2, D19, P34,W92, Y90, A39, V5, T123 V3, Q2, D19, P34, W92, Y90, A39, V18, T91 Epi#45K77, H74, F75, N71, D69, G67 K77, H74, F75, N71, D69, V76 K77, H74, F75,N71, D69, V65 Epi#46 A98, R128, R31, P95, N93, G32 A98, R128, R31, P34,G20, Q2 Epi#48 Q2, D19, P34, P95, G32 H30, K96, P95, P34, G20 Epi#49D87, D42, L40, Q85, Q84, C78, T47, Q45, K48 D87, D42, L40, Q85, Q84,C78, T47, Q45, K82 Epi#50 D19, W92, P34, T91 D19, W92, P34, P95 D19,W92, T91, T36 Epi#51 D129, H30, K33, R31, R128, K126, H124 R31, H30,D129, R128, K100, K126, H124 T123, H124, K126, R128, R31, K33, H30Derp2: Epi#03 L17, K89, A39, I13, Y86 L17, K89, A72, I88, Y90 L17, K89,A72, I52, Y90 Epi#04 K15, S1, Q2, K14, V16, L17 K15, S1, Q2, K14, A39,L17 K15, S1, Q2, K14, V40, I13 Epi#05 G60, A56, L61, P99, G32, R31, H30,I97 G60, A56, L61, P99, G32, R31, H30, I28 Epi#06 G60, A56, D64, S57,K55, P66 G83, N46, D114, T49, K48, P79 G60, N103, D59, S101, R31, P95Epi#08 K55, D64, S57, V106, F35 K55, E62, S57, V106, F35 Epi#09 L61,G60, E102, R128, 128, K126, N103, T123, V105 L61, G60, E102, R128, 1127,K100, N103, T123, V105 L61, G60, E102, R128, 1127, H124, N103, T123,V105 Epi#10 SAS: 435, Size 24.47: D69, T91, N93, F35, G32, R31 SAS: 422,Size 20.74: E38, T91, N93, F35, G32, K96 Epi#11 K14, I13, Q85, V81, E42K15, I13, Q85, V81, E42 K14, I13, Q85, V40, D87 Epi#12 Y86, E42 Y90, E53Y90, E38 Epi#13 H30, A125, P26, T123, A122, P19, L37, P34, W92 H30,A125, P26, T123, A122, H124, S24, G23, G20 H30, A125, P26, T123, A122,P19, L17, G20, F35 Epi#15 K55, P66, D69, I68, K89, A72 K55, P66, D69,I68, K89, A39 K55, P66, D64, I54, K109, G115 K55, P66, D64, I54, K109,A9 Epi#18 R31, 129, A125, S101, E102, N103 R31, 129, A125, S101, E102,V104 R31, 129, A125, T123, A122, V105 Epi#22 D69, P66, D64, V65, K55D64, P66, D69, T91, K89 D59, L61, D64, P66, W92 D59, L61, D64, V65, E62D69, P66, D64, V65, E53 Epi#24 D64, K55, E62, P99, R31, P34, K96 E53,K55, E62, P99, R31, P95, K96 D64, K55, E62, P99, R31, A98, K96 Epi#25R31, H30, I28, E102, N103 R128, K126, I127, E102, N103 R128, K126, I28,E102, V105 Epi#27 D64, E53, D69, K89 D69, E53, D64, K55 D59, E62, D64,K55 Epi#28 V40, D87, Q85, E42, Q84, G83, K82 G20, H22, Q2, L17, E38,L37, Q36, P34, K33 G20, H22, Q2, L17, E38, L37, F35, P34, K33 Epi#29I97, K100, L61, E62 G60, N103, L61, E62 I127, N103, L61, E62 Epi#30 G60,N103, S101, H30, K96, I97, P95 G60, N103, A125, H30, K96, I97, P95 I28,I127, A125, H30, K96, I97, P95 Epi#33 Q36, F35, V106, S57, A56, K55 K33,F35, V106, S57, A56, K55 Epi#34 I28, P26, S24, G23, G20, T123, S57 I28,P26, S24, V3, G20, T123, T107 W92, P34, T91, V18, G20, T123, P26 Epi#37P66, V63, L61, K100, N103 P95, A98, L61, K100, N103 P19, V18, L17, K89,D87 P19, V3, L17, K89, D87 T123, V104, L61, K100, N103 Epi#38 L61, G60,E102, A125, V105, N103, P99, S57 L61, G60, E62, A56, V105, N103, P99,S57 Epi#39 A125, E102, H124, T123, P26, G20, L17 Epi#40 G60, L61, A56,T107, K6, T123 A39, L17, G20, T123, P26, S24 G60, L61, A56, T107, K55,S57 G60, L61, A56, T123, K126, S101 Epi#41 P19, L17, V3, S1 P19, L17,V5, S24 Epi#44 V65, D64, P66, W92, Y90, A39, V18, P19 L61, D64, P66,W92, Y90, A39, V18, T91 Epi#45 R31, P34, F35, N93, V94 K96, P34, F35,N93, G32 Epi#47 I127, S101, R31, 197, A98, L61, N103, P99, P95 I28,S101, R31, 197, A98, L61, N103, P99, S57 Epi#48 H30, K96, P95, P99, G60H30, K96, P34, P19, G20 H30, K96, P34, P19, V18 H30, K96, P34, P95, V94H30, K96, P34, P19, V3 E38, K89, P70, P66, V65 H30, K96, P95, P34, G32Q36, K89, P70, P66, V65 Epi#50 D69, Y90, W92, P66, P70 D69, Y90, W92,P34, P95 D69, Y90, W92, T91, P34 D69, Y90, W92, V94, P95 D69, Y90, W92,L37, P19 Epi#51 K126, H124, E102, R128, 128, R31, H30 T123, H124, K126,R128, 128, R31, H30 D4, H124, K126, R128, 128, R31, H30

PhIp2:

Epi#02 T87, K85, Q61, S38, R34, R67 T87, K85, Q61, P63, R34, V42 Epi#03K10, A90, I88, Y86 K10, A18, I88, Y86 Epi#04 R34, S38, Q61, K85, T87,I88 R34, S38, Q61, K85, T87, A90 Epi#05 G47, A18, S12, T87, G89, T91,T5, V1 G73, A29, L69, T27, G50, T53, T45, V42 G11, A18, L20, T91, G89,A90, T87, I88 Epi#06 A93, P94, D79, R34, Q61, P59 A93, P94, D79, R34,Q61, P83 A93, P94, D80, R34, Q61, P59 A93, P94, D79, R34, Q61, P63Epi#08 K10, E9, G11, A18, H16, F54 K46, E48, G47, A18, H16, F54 K10, E9,S12, A18, H16, F54 Epi#09 L69, T27, G73, N76, R67, V77, D79, R34, A43,T45, V42 L69, T27, A29, E30, R67, V77, D80, R34, A43, T45, V42 Epi#10D55, A18, N13, S12, F54, G47, K46 T45, A18, N13, S56, F54, G47, K46Epi#09 L60, S56, E57, D55, K15, N13, S12, G11 L60, S56, E57, D55, H16,F54, T45, T53 L60, S56, E57, D55, H16, F54, T45, G47 Epi#12 Y86, E84Y23, E24 Epi#18 N76, R67, F78, V81, A93, Y92, T91, T5, P2, V1 Epi#19D39, W41, S38, Q61, R34, G37 E40, W41, S38, Q61, R34, A43 Epi#22 D79,P94, D80, P83, K85 D79, P94, D80, P63, K85 Epi#23 K10, N13, E14, L60,Q61, P59 K10, N13, E14, L60, Q61, P83 K10, N13, E14, L60, Q61, P63Epi#24 E58, K15, E57, P59, S56, E14, Q61 D55, K15, E57, P59, S56, E58,Q61 Epi#25 R34, R67, W41, D39, E40 Epi#26 S38, E40, W41, V42, E32, E30S38, E40, W41, V42, A43, E32 Epi#27 E14, E57, E58, K15 D55, E14, E84,K85 Epi#28 G37, H36, Q61, K85, E84, L60, F54, A43, K46 G37, H36, Q61,K85, E84, L60, F54, S12, D55 G37, H36, Q61, K85, E84, L60, F54, S56, D55G37, H36, Q61, K85, E84, L60, F54, A43, R67 G37, H36, Q61, K15, E57,L60, F54, A43, K46 G37, H36, Q61, K85, E84, L60, F54, S12, K15 G37, H36,Q61, K85, E84, L60, F54, S56, K15 G37, H36, Q61, K85, E84, L60, F54,A43, R34 G37, H36, Q61, K85, E84, L60, F54, A18, D55 Epi#29 G73, K72,L69, R67, E30 I88, N13, L60, F54, E57 G25, K72, L69, R67, E32 V77, K75,L69, R67, E30 G37, H36, L60, F54, E57 G37, Q61, L60, F54, E57 Epi#30I88, N13, S12, H16, K15, P59, L60 I88, N13, S56, H16, K15, L60, P59 I88,N13, A18, H16, K15, P59, L60 Epi#33 K46, F54, V42, S56, K15 H16, F54,V42, S56, K15 Epi#34 V1, P2, T5, V4, P94, Y92, T87 V1, P2, T5, L20, G89,T91, T87 V81, P94, T5, V1, P2, Y92, T91 Epi#37 T27, A29, L69, K72, D26A43, R67, L69, K75, N76 Epi#38 L20, G89, E9, A18, N13, P59, S56 Epi#40G49, L20, G89, Y86, K85, T87 G49, L20, G89, T87, K10, S12 G49, L20, G89,T87, K10, T7 Epi#44 V77, R67, D79, P94, Y92, A93, V1, P2 L69, R67, D79,P94, Y92, A93, V1, T5 Epi#45 D79, P94, F78, N76, M74, L69 D80, P94, F78,R67, D79, V77 K3, P94, F78, N76, M74, G73 Epi#46 A43, R67, R34, P63,H36, Q61 V77, R67, R34, P63, H36, G37 L69, R67, R34, P63, G37, Q61Epi#47 G37, E35, E40, A43, R34, L60, N13, P59, S56 V77, E32, E40, A43,R34, L60, N13, P59, S56 S38, G37, E40, A43, R34, L60, N13, P59, S56Epi#48 E24, K3, P94, P2, V1 E84, D80, P94, P2, V1 Epi#50 D39, W41, A43,T45 D39, W41, V42, T45 Epi#51 D79, H36, E84, T87, K10, G11, H16 D39,H36, Q61, K85, P63, R34, W41 D79, H36, E40, D39, G37, R34, W41 Q61, H36,E84, T87, K10, G11, H16 Example 11

For this example a third-generation epitope sequences were determinedfor some additional enzymes and redetermined for all of the enzymes inexample 1-3. New enzymes are AMG (AMG pdb), BPN″ (1sup.pdb), Esperase(structure see Appendix D), Natalase (structure modelling based onSP722), Amylase-AA560 (Structure modelling based on SP722), Protease A,Alcalase, Protease B, ProteaseC, ProteaseD, ProteaseE, Properase andRelase based on their sequences and structures. The structures ofProtease B, Properase, Relase, Protease A, Alcalase, ProteaseC,ProteaseD and ProteaseE can be found by “Homology modelling” (see above)and computer modelling of the epiope patterns that had been assembled inour database (shown in Table 8). Furthermore, the epitope sequences wereredetermined for CAREZYME, Laccase, PD498, Savinase, Amylase SP722, andCellulase, according to the method.

The protein surface is scanned for epitope patterns matching the given“consensus” sequence of about 6-12 residues. First, residues on theprotein surface that match the first residue of the consensus sequenceare identified. Within a specified distance from each of these, residueson the protein surface that match the next residue of the consensussequence are identified. This procedure is repeated for the remainingresidues of the consensus sequence. The method is further describedunder the paragraph “Methods” above and the program can be found inAppendixes.

The critical parameters used in this screening included:

-   -   i) a maximal distance between the alpha-carbon atoms of        subsequent amino acids,    -   ii) a minimal accessability of the amino acid of 20 Å2,    -   iii) the largest maximal distance between the most distinct        amino acids should be less than 25 Å    -   iv) the best epitope were taken,    -   v) the homology with the epitope pattern of interest was 100%

In this way a number of potential epitopes are identified. The epitopesare sorted according to total surface accessible area, and certainentries removed:

-   -   1) Epitopes that contain the same protein surface residue more        than once. These are artefacts generated by the described        algorithm.    -   2) Epitopes which are “too big”, i.e. where a distance between        any two residues in the epitope exceeds a given threshold.

The subtilisin sequences and positions mentioned in the following arenot given in the BPN′ numeration but in the subtilisins own numeration(see the alignment as described above in Tables 1A and 1B).

The epitope sequences found were:

AMG: Epi#01 L104, P123, P107, R125, R122, N182, S184, Q172, T173 L104,P107, P123, R125, R122, N182, S184, Q172, S453 L104, P107, P123, R125,R122, N182, S184, Q172, T452 Epi#02 L234, R241, S240, F237, T173, Y175,R122, R125 L234, R241, S240, F237, T173, Y169, R125, R122 L234, R241,S240, F237, T173, Y175, R125, R54 Epi#03 L291, K404, I288, Y289 L66,K61, H254, I253, Y329 Epi#04 R122, Y175, S184, Q172, Y169, A454, I455R122, Y175, S184, Q172, Y169, N171, A451 R125, Y175, S184, Q172, Y169,T452, A451 Epi#06 G31, A24, D25, S30, A27, P41 G146, N145, D144, T148,S149, P467 A471, N145, D144, T148, S149, P467 Epi#07 G294, T290, S405,D293, S287, R286, P307, D283 G294, T290, S287, D293, S296, R286, P307,D283 G207, T204, S200, D214, S209, R160, P157, D153 G294, T290, S405,D293, S287, R286, P307, D309 Epi#08 A27, D25, S30, V111, F49 A24, D25,S30, V111, F49 Epi#09 S149, T148, G146, N145, A471, R68, N69, T72, V470S73, S76, T72, N69, R68, A471, N145, T148 Epi#10 D238, N182, N236, S240,F237, R241, K244 D238, T173, N182, S239, F237, R241, K244 Epi#11 F49,F109,191, Q85, E113 Epi#12 Y363, E342 Y311, E308 Y175, E180 Epi#13 S119,W120, P123, A102, P94, S92, G90, L98 S119, W120, P123, A102, P94, S92,G96, G90 Epi#15 K244, P307, D283, I288, T290, G294 R160, P157, D153,I154, T462, G90 R286, P307, D283, I288, T290, G294 Epi#16 L410, P46,Y48, R413, S397, S394, A392, A393, N395 R160, P157, Y458, G456, S211,S209, A205, A201, D214 Epi#17 A201, S209, R160, S459 A205, S209, R160,S459 Epi#19 D44, N45, S411, Q409, R413, L410 D47, N45, S411, Q409, R413,L410 Epi#20 K61, P434, L66, L423, N427, D65, G70, D71 Epi#22 D357, S356,D349, V346, D345 D349, S356, D357, A359, D345 D357, S356, D349, L348,D345 Epi#23 K404, N292, E299, S298, L295, A300 K404, N292, E299, S296,L295, A300 Epi#24 D336, K337, E259, P258, S431, L332, K378 D336, K337,E259, P258, S431, R429, K378 D336, K337, A261, P258, S436, E259, Q338Epi#25 R125, R122, W120, E180, N182 R241, K244, E308, N313 Epi#26 W212,S200, E198, W437, V197, G438, E259 W212, S200, E198, W437, V197, A201,D214 Epi#27 D283, E280, D349, K352 D403, E408, D406, K404 D349, E280,D283, K244 D349, E280, D283, K279 Epi#28 L332, D336, Q338, K337, E259,C262, P272, D345 V374, D336, Q338, K337, E259, C262, P272, D345 G339,D336, Q338, K337, E259, C262, P272, D345 Epi#29 L295, G294, L291, R286,E299 I288, K404, L291, R286, E299 L348, K352, L354, F380, E299 Epi#33K352, Y355, V374, S371, S365, K337 K352, Y355, V374, S365, S340, K337Epi#34 V463, W466, S468, V470, P467, T464, T462 I469, W466, S468, V470,P467, T464, T462 I154, W466, S468, V470, P467, T464, T462 V463, W466,S468, V470, P467, S465, T464 Epi#37 T362, A359, L348, K352, D357 T360,V346, L348, K352, D357 T362, A359, L348, K352, D349 Epi#38 G438, E259,A435, R68, L66, N69, P434, S431 Epi#39 A353, E299, R286, P307, G243,L234 A300, E299, R286, P307, G243, L234 Epi#40 A205, L143, G146, Y147,P467, T464 G146, L143, A205, T204, A201, S209 A451, A450, T448, P446,S444 Epi#41 P467, Y147, L143, V206, S149 Epi#42 L66, P434, S431, N430,R429, R428 L104, P123, S95, G101, P94, R122, R125 L104, P107, S95, G96,P123, R125, Q172 Epi#44 L143, Q140, D144, W141, Y147, S468, V470, T72V206, Q140, D144, W141, Y147, S468, V470, P467 S211, Q216, D214, P218,Y223, A451, A450, T448 S211, Q216, D214, P218, Y223, A450, G447, T448Epi#45 R413, P46, F49, Y50, N110, D112, G31 R413, P41, F49, Y50, N110,D33, G31 D44, P46, F49, Y50, N110, D112, G31 Epi#46 Y175, R125, R122,P123, G174, Q172 Y169, R125, R122, P123, G174, Q172 V432, R429, R428,P434, N69, G70 Y175, R125, R122, P94, N93, G90 Y175, R122, R125, P123,N182, G121 Y175, R125, R122, P94, G101, A102 Y175, R125, R122, P94,G118, A115 Y175, R125, R122, P94, G101, G96 Y175, R122, R125, P123,N182, G183 Epi#48 S211, D214, P218, P446, G447 E259, K337, P258, P434,V432 S215, D214, P218, P446, G447 S209, D214, P218, P446, V445 E259,K337, P258, P434, V433 Epi#50 R122, Y175, W120, T117, S119 R125, Y175,W120, S119, T117 Epi#51 T390, H391, E408, Q409, R413, S411, W317 T390,H391, E408, S405, I288, K404, W317 D406, H391, E408, Q409, R413, S411,W317 T390, H391, E408, D406, K404, Q409, W317 Epi#52 W437, A260, T266,R273, W228, D264, Q225 BPN′: Epi#02 T255, K256, S260, F261, P194, Y262,R186, V203 L257, K256, S260, F261, P194, Y262, R186, V203 T253, K256,S260, F261, P194, Y262, R186, V203 Epi#03 K141, A137, I108, Y104 K136,A137, I108, Y104 K136, A134, I108, Y104 Epi#04 K265, Y262, S188, Q185,R186, N184, L257 K265, Y262, S188, Q185, Y263, R186, L257 K265, Y262,S188, Q185, R186, N184, G258 K265, Y262, S188, Q185, Y263, R186, G258Epi#05 G80, A1, N77, P40, G211, S38, S37, V44 G80, A1, N77, P40, G211,S38, S37, L42 G127, A152, N155, T164, G160, S158, S188, Y262 Epi#06G211, N212, D36, S37, K43, P40 G80, N212, D36, S38, K43, P40 G211, N212,D36, S38, K43, P86 Epi#08 K256, D259, S260, F261 K43, D36, S38, V44, F58Epi#09 S105, S132, A133, A137, D140, K141, A144, S145, N118 S248, T244,A144, S145, D120, K27, N118, A116, N117 Epi#10 E54, T55, N57, S37, F58,G46, K43 T55, A48, N57, S37, F58, G46, K43 E54, T55, N57, S49, F58, G46,K43 Epi#11 K136, I108, Q103, V51, D98 Epi#12 Y171, E195 Epi#13 S101,W106, P52, T55, A48, P56, S49, G47, F58 S105, W106, P52, T55, A48, P56,S49, G47, W113 Epi#15 N25, P239, D120, I115, K141, A144 N240, P239,D120, I115, K141, A144 Epi#16 Q271, P14, Y21, G20, Q19, S18, A15, A272,N252 Q59, P210, Y214, G211, S38, D36, D61, A99, D98 Epi#17 A187, S188,R186, S183 A187, S188, R186, S182 Epi#18 N184, R186, S188, G157, S158,T159, S161 N184, R186, S188, G157, S158, T159, S162 N184, R186, S188,G157, S158, E156, N155 N184, R186, S188, G157, S158, E156, F189 Epi19E156, N155, S188, Q185, R186, L257 E156, N155, S188, Q185, R186, G258E156, N155, S188, Q185, R186, A187 Epi#22 D197, S260, D259, L257, K256D197, S260, D259, Y263, K256 Epi#23 N155, E156, S188, Q185, A187 Epi#24E156, G166, E195, P194, S260, L257, K256 D259, G264, E195, P194, S260,L257, K256 D197, K170, E195, P194, S260, L257, K256 Epi#25 K141, I115,D120, N25 K141, I115, D120, N118 K141, I115, E112, N118 Epi#26 W113,S49, W106, P52, E54, D98 W113, S49, W106, P52, E54, D60 W113, S49, W106,V51, E54, D98 Epi#28 A99, D61, Q59, F58, E54, L96, Q103, G102, D98 A99,D98, Q59, F58, E54, L96, Q103, G100, D61 A99, D61, Q59, F58, E54, L96,Q103, S101, D98 Epi#29 G102, Q103, L96, E54 G100, Q103, L96, E54 Epi#30I79, N76, S87, H17, S18, P14, V4 I79, N76, S87, H17, Q19, P14, V4 Epi#31L257, Q185, N184, R186, F189, V203, I205, D181 L267, Q10, N184, R186,F189, V203, I205, D181 Epi#33 K213, Y214, P210, S38, S37, K43 Q59, F58,V44, S38, S37, K43 Epi#34 W106, P52, M50, G47, P56, T55, S53 W106, P52,S49, G47, P56, T55, S53 I115, W113, M50, V51, P52, T55, S53 I108, W106,S105, V51, P52, T55, S53 Epi#35 A99, L96, S49, M50, 1108 A99, L96, S49,M50, 1107 Epi#36 A137, A134, A133, G131, Y104, S105, Q103, V51, A48,W113 A134, A137, A133, G131, Y104, S101, Q103, V51, A48, W113 Epi#37Y262, R186, L257, K256, D259 Y263, R186, L257, K256, N252 Epi#39 E156,T164, P129, G127, L126 E156, T164, P129, G128, L126 E156, T164, P129,G154, L126 E156, T164, P129, G166, L126 Epi#40 R247, L250, A272, T255,K256, S260 R186, L257, G258, Y263, K256, S260 G264, L257, G258, T255,K256, S260 Epi#41 P194, Y262, L257, S260 P194, Y263, L257, S260 Epi#42P194, S260, G258, R186, Q185 Epi#44 S182, Q185, D181, Y6, S9, V4, P14S183, Q185, D181, Y6, S3, V4, P5 S248, R247, D197, P194, Y262, S260,G258, T255 S53, P52, W106, Y104, S105, V51, T55 Epi#45 K170, P194, F261,Y262, R186, D181, V203 D197, P194, F261, Y262, R186, D181, V203 Epi#46S162, S158, E156, N155, A187, Q185, N184, R186, S188 S188, S158, E156,N155, A187, Q185, N184, R186, S183 S158, S188, E156, N155, A187, Q185,N184, R186, S182 S161, S158, E156, N155, A187, Q185, N184, R186, S183G160, S158, E156, N155, A187, Q185, N184, R186, S188 Epi#48 S38, K43,P40, P210, G211 S37, K43, P86, P14, V4 S38, K43, P40, P210, G215 Epi#50H238, W241, T242, P239 H238, W241, T244, T242 H238, W241, T242, T244Epi#51 T242, H238, Q275, Q271, P14, S18, H17 Q245, H238, Q275, K237,P239, T242, W241 Q275, H238, Q245, T242, R247, T244, W241 Q245, H238,Q275, Q271, P14, Q19, H17 CAREZYME Core: Epi#01 P61, P165, K164, R158,N154, Y168, R153, S151 P137, P49, K44, K13, N32, Y54, Q36, T39 P61,P165, K164, R158, N154, S152, R153, S151 Epi#02 L115, N118, S117, R4,T6, Y147, R146, V129 L115, N118, S5, R4, T6, Y147, R146, V129 Epi#03K44, A43, I38, Y54 K13, A43, I38, Y54 Epi#04 R153, S151, Q145, Y147,R146, 1131 R153, S151, Q145, Y147, R146, G144 R153, S151, Q145, Y147,R146, L142 Epi#05 G3, A1, S183, T95, G101, A100, S96, G97 G3, A1, F184,T93, G101, T95, S96, G97 G97, A100, S96, T95, G101, T93, S183, G3 Epi#06G140, P160, D161, R158, K164, P165 G50, P137, D133, R146, Q145, P143A162, P165, D161, R158, K164, P160 Epi#07 G148, T6, S181, D178, R170,P165, D58 G128, T6, S181, D178, R170, P165, D58 Epi#08 K44, D42, S45,A43, F41 Epi#09 A191, E192, R196, A195, R200, N25, N202, N₂O₆ D161,R158, D157, R153, N176, S151, N154 Epi#10 D161, A57, N34, A162, F159,R158, K164 D2, A1, R185, S183, F184, G3, R4 Epi#11 F41, F29, I38, Q36,D58 Epi#12 Y168, E155 Y90, E91 Epi#13 A63, W62, P165, T60, A162, P160,L142, G149, Y147 A63, W62, P165, T60, A162, P160, L142, G128, Y147 A63,W169, P165, T60, A162, P160, L142, G144, Y147 Epi#15 P137, D133, I131,R146, G144 P137, D133, I131, R146, G148 P137, D133, I131, R146, G130P137, D133, I131, R146, G128 P137, D133, I131, R146, G149 Epi#16 Q138,P137, Y54, R37, Q36, N34, A162, A57, D161 R170, P165, Y168, R153, S151,N176, D172, A63, D67 R170, P165, Y168, R153, S151, N176, D172, A63, D66Epi#17 A1, S183, R4, S117 A100, S181, R4, S183 A1, S183, R4, S5 Epi#18N118, R4, S181, - - - , G3, - - - , S117, L115, - - - , A78, S80 N34,N32, R37, F35, - - - , A33, Y54, S45, - - - , - - - , A43, V52 Epi#19D157, N154, S151, Q145, R146, L142 D178, N176, S151, Q145, R146, G144Epi#22 D40, A43, D42, W18, K20 D40, A43, D42, A19, K20 Epi#23 R158,N154, E155, L142, Q145, P143 R153, N154, E155, S151, Q145, P143 Epi#24D42, K44, E48, P137, F139, A33, Q36 D40, K44, E48, P137, F139, A33, Q36D161, K164, A162, P160, R158, L142, Q145 D161, K164, E155, P143, R158,L142, Q145 Epi#25 R158, K164, W169, D172, N176 R4, H119, 177, E82, N81Epi#26 W18, S15, E82, W85, P23, A19, D42 W18, S15, E82, W85, P23, G84,D203 Epi#28 I31, D133, Q138, L142, E155, K164, F159, P165, D161 I131,D133, Q138, L142, E155, K164, F159, P143, R158 I131, D133, Q138, L142,E155, K164, F159, P160, R158 Epi#29 I131, R146, L142, R158, E155 G144,Q145, L142, R158, E155 Epi#30 G79, N81, A78, H119, S117, I77, L115 G79,N81, A78, H119, S76, I77, L115 Epi#31 L142, R158, N154, R153, W169,F171, D172 Epi#33 Q36, F29, P27, S15, A19, K20 K44, F41, P27, S15, A19,K20 Epi#34 V129, P143, S151, G144, R146, Y147, T6 V129, P143, S151,G148, R146, Y147, T6 V129, P143, S151, G149, R146, Y147, T6 Epi#36 A83,A22, A19, S15, K13, V52, A43, W18 Epi#37 Y147, R146, L142, R158, D161Y147, R146, L142, R158, N154 Y147, R146, L142, R158, D157 Epi#38 E155,R158, P160, G140, L142 E155, R158, P143, G144, L142 Epi#40 G79, L115,G113, T111, A74, T6 G79, L115, G113, T111, A74, S15 G79, L115, G113,T111, A74, S110 G116, L115, G113, T111, A74, T6 G79, L115, G113, T111,A74, S76 Epi#42 L142, P143, S151, G144, R146, Q145 L142, P143, S151,G148, R146, Q145 L142, P143, S151, G149, R146, Q145 Epi#44 L142, R158,D161, P165, W62, Y168, S152, G144, P143 I131, R146, D133, P137, Y54,A33, V52, P49 L142, R158, D161, P165, W62, Y168, S152, G149, P143 Epi#45R185, P208, F207, N206, D203, V24 D67, P213, F68, N65, D66, V64 R185,P208, F207, N206, D204, G205 Epi#46 A195, R200, R201, P23, N202, G205A191, R200, R201, P23, N202, G205 V24, R201, R200, P190, Q211, A209Epi#47 A191, A195, E192, V194, R200, N202, R201, P23 A195, A191, E192,V194, R200, N25, R201, P23 A191, A195, R196, V194, R200, N202, R201, P23Epi#48 E48, K44, P49, P137, V52 E48, K44, P49, P137, G50 E48, K44, P49,P137, G140 Epi#50 D172, Y168, W62, V64, P213 D42, W18, A43, T39 D67,W173, W62, V64, P213 D66, W173, W62, V64, P213 D42, W18, S45, P49 D172,W169, W62, V64, P213 Epi#51 R4, H119, D2, T95, P98, K175, W169 R4, H119,D2, R185, P208, Q186, W85 R4, H119, D2, T95, G97, K175, W173 Epi#52 W18,A22, R200, R201, W85, Q186 Esperase: Epi#01 N24, P239, R237, K235, N243,S240, Q245, T242 N24, P239, K235, R27, N117, Y91, R43, S87 N24, P239,R237, K235, N243, Y241, Q245, S240 Epi#02 T3, N76, L75, R43, S38, Y209,R213, V215 T3, N76, S87, R43, S38, Y209, R213, V215 T129, N166, Q161,R160, T156, Y192, R186, V203 Epi#03 R186, Y192, S261, Q161, R160, N155,G127 R186, Y192, S261, Q161, R160, N155, G157 R186, Y192, S261, Q161,R160, N155, L126 R186, Y192, S261, Q161, R160, T156, G162 R186, Y192,S261, Q161, R160, N155, A187 Epi#05 G102, A105, S133, T134, G131, R170,T129, Y167 G102, A105, S133, T134, G131, R170, T129, G127 G211, A37,R43, P40, G80, T3, S78, I79 Epi#06 G211, N61, D97, R98, S53, P55 G102,N99, D97, R98, S53, P55 G100, N99, D97, R98, S53, P55 Epi#07 211, T210,D60, S38, R43, P86, D89 Epi#08 A108, E136, S133, A105, F50 A108, E136,S132, A105, F50 A187, D181, S188, V203, F189 Epi#09 N212, G211, S38,H59, N61, N99, R98 S52, S53, R98, N99, N61, G211 Epi#10 T129, T156,N155, S188, F189, G157, R160 D181, N183, R186, S188, F189, G157, R160T129, N166, N155, S188, F189, G157, R160 T129, T156, N155, S218, F189,G157, R160 D97, N99, N61, S57, F50, G102, R98 Epi#12 Y167, E136 Y192,E195 Y171, E136 Epi#13 S38, R43, P40, A37, H59, S57, P55, Y58 S38, R43,P40, A37, H59, S57, P55, F50 S38, R43, P40, A37, H59, S49, P55, Y58Epi#15 N24, P86, D89, I44, R43, A45 N24, P86, D89, I44, R43, G46 N76,P86, D89, I44, R43, A45 N24, P86, D89, I44, R43, A37 Epi#16 Q161, P194,Y192, G157, R160, S188, D181, A187, N183 Q161, P194, Y192, R186, Q185,S188, D181, A187, N183 Q161, P194, Y192, G162, R160, S188, D181, A187,N155 Epi#17 A37, S38, R43, S87 Epi#18 N144, N140, R141, L137, S133,T134, E136, S132 N140, N144, R141, L137, S133, T134, A105, S103 N143,N144, R141, L137, S133, T134, E136, N140 Epi#19 I21, N18, Q15, Q275,R19, G20 I21, N18, Q15, Q275, R237, G20 E197, N265, S261, Q161, R160,G162 E197, N265, S261, Q161, R160, G157 I21, N18, Q15, Q275, R237, G25Epi#23 R98, N61, E54, S53, F50, P55 R98, N61, E54, Y58, F50, P55 R98,N61, E54, S57, F50, P55 R98, N61, E54, S52, F50, A105 Epi#24 E195, G264,E197, P260, S261, P194, Q161 D89, G46, A48, P55, S52, F50, Q109 E197,G264, E195, P194, S261, L262, Q161 Epi#25 R98, H59, E54, N61 R98, H59,D60, N61 R43, H39, I44, D89, N24 R27, H120,1115, E112, N116 Epi#28 L104,Q109, I115, E112, W113, F50, S53, R98 A105, Q109, I115, E112, W113, F50,G102, R98 A108, Q109, I115, E112, W113, F50, S53, R98 V107, Q109, I115,E112, W113, F50, S53, R98 Epi#29 I147, N140, L137, R141, E136 G146,N140, L137, R141, E112 I115, N143, L137, R141, E136 G102, N99, L96, R98,E54 Epi#30 G211, N212, S38, H59, S57, I51, P55 G211, N61, S57, H59, S38,P40, L75 G211, N212, S38, H59, S49, I51, P55 G211, N212, S38, H59, P55,I51, L96 Epi#31 L257, Q185, N183, R186, F189, V203, D181 L262, Q185,N183, R186, F189, V203, D181 Epi#33 H59, Y58, P55, S52, S53, R98 Q109,F50, P55, S57, S53, R98 Q109, F50, P55, S49, S53, R98 Epi#34 I79, P40,S38, G211, R213, Y209, S216 I79, P40, S38, G211, R213, Y214, T210 I51,P55, S49, L96, R98, S53, S52 Epi#37 T134, A108, L137, R141, N144 Y256,A254, L257, R186, N183 A105, A108, L137, R141, N144 Epi#38 L257, G264,E195, L262, N265, P260, S259 L257, G264, E195, L262, N265, P260, S261Epi#39 E195, R170, P194, G264, L257 E195, R170, P194, G264, L262 Epi#40R141, L137, A108, T134, A105, S133 R43, L42, A37, Y58, P55, S52 R186,L257, A254, Y256, P260, S259 R186, L262, G258, Y256, P260, S259 R186,L257, G184, Y256, P260, S259 R141, L137, A108, T134, A105, S103 R186,L262, G264, Y256, P260, S259 R186, L257, A254, Y256, P260, S261 R186,L262, G258, Y256, P260, S261 R186, L257, G264, Y256, P260, S261 Epi#41P260, Y256, L257, S259 Epi#42 L75, P86, S87, N24, P239, R237, Q275 L75,P86, S87, N24, P239, R237, R19 Epi#44 S53, R98, D97, Y58, S57, A48, P55S53, R98, D97, Y58, S38, G211, T210 Epi#45 R19, H17, F22, N24, D89, G25R43, P86, F22, N24, D89, G25 R272, H269, F10, N183, D181, V203 R272,H269, F10, N183, D181, G184 R43, P86, F22, N24, D89, G46 Epi#46 R19,R237, P239, N24, G20 R19, R237, P239, N24, G25 Epi#47 G162, Y192, R160,N155, A187, Q185, N183, R186, S188 G157, Y192, R160, N155, A187, Q185,N183, R186, S188 S261, Y192, R160, N155, A187, Q182, N183, R186, S188L262, Y192, R160, N155, A187, Q182, N183, R186, S188 Epi#48 S261, Q161,P194, P260, G258 S261, Q161, P194, P260, G264 Epi#50 D181, W6, V4, T3D181, W6, V203, S188 D181, W6, V4, S9 D181, W6, T3, P5 Epi#51 R98, H64,T210, R213, P40, S38, H59 R98, H64, T210, R213, G211, S38, H59 R19, H17,Q15, Q275, R272, Q252, H269 Laccase: Epi#02 A14, N15, S17, F21, P180,Y176, R266, V177 T22, N15, P18, F21, P180, Y176, R266, V177 A274, N275,A181, R175, P180, Y176, R266, V177 A24, N15, S17, F21, P180, Y176, R266,V177 T272, N275, A181, R175, P180, Y176, R266, V177 Epi#03 L184, K173,I186, Y256 Epi#04 R234, S211, Q261, K264, N267, G271 R234, S211, Q261,K264, R266, G268, R259, S211, Q302, R234, N299, A301 R259, S211, Q236,R234, N299, A301 Epi#05 G372, A371, L369, P350, G81, S349, S351, V352G372, A371, L369, P350, G81, S351, S349, Y347 Epi#06 G286, N289, D291,T293, S295, P292 G214, P252, D254, T293, S295, P298 A288, N289, D291,T293, S295, P292 Epi#07 G214, T294, D291, R283, V253, P252, D254 G30,T12, D53, R59, A497, P89, D51 G30, T10, D51, R59, A497, P55, D53 Epi#08A371, E348, S349, A346, F335 A14, D53, G90, A92, H91, F93 A181, E183,G20, V16, F21 A181, E183, G20, A182, F21 Epi#09 N41, A100, N43, V6, D42,R37, N4, T8, L94 N41, A100, N43, V6, D42, R37, N4, T8, N47 L369, N366,E376, R379, N472, A471, V474 Epi#10 E183, A181, N275, T272, F273, G268,R266 D129, N41, N43, A100, F69, G72, R71 E183, A181, N275, A274, F273,G271, K264 Epi#11 F93, L486, I489, Q485, V481, E482 Epi#12 Y490, E488Y375, E376 Epi#13 N366, P370, D367, I358, Q363, A471 N366, P370, D367,I358, Q363, G361 R379, P378, D326, I319, T321, G323 R379, P378, D326,I319, T321, G318 R379, P378, D326, I319, T321, A324 Epi#15 N366, P370,D367, I358, Q363, A471 N366, P370, D367, I358, Q363, G361 R379, P378,D326, I319, T321, G323 R379, P378, D326, I319, T321, G318 R379, P378,D326, I319, T321, A324 Epi#16 R175, P180, Y176, R266, Q164, N267, D166,A163, D205 R283, P292, Y256, G214, Q251, D254, A285, A288, N289 R283,P292, Y256, G214, Q251, D254, D291, A290, N289 Epi#17 A306, S413, R409,S414 A411, S413, R409, S414 A306, S410, R409, S414 A411, S414, R409,S410 Epi#19 E216, N250, Q251, Q191, R283, G286 E190, N250, Q251, Q191,R283, A288 E216, N250, Q251, Q191, R283, A290 E190, N250, Q251, Q191,R283, A285 Epi#22 D491, P494, D492, P495, E496 D492, P494, D491, L493,E496 Epi#23 R339, N460, E348, S349, L369, A371 R339, N460, E348, S351,L369, P370 R339, N460, E348, S351, L369, A365 R339, N460, E348, S351,L369, P350 R283, N188, E190, N250, Q191, P252 Epi#24 D475, G72, A476,P445, R379, A471, Q363 D53, G90, A497, P495, T498, P55, Q501 D53, G90,A497, P495, S499, L58, Q501 Epi#25 R37, K40, D129, N130 R37, K40, D129,N41 Epi#27 E142, E139, D138, K194, E142, E139, D138, K193 Epi#28 L58,Q501, I500, E496, L493, P495, D492 G286, D254, Q191, K194, E190, K193,G192, D138 A288, D254, Q191, K193, E190, K194, G192, D138 G192, D248,Q191, K194, E139, L136, A135, D138 V253, D254, Q191, K193, E190, K194,G192, D138 A285, D254, Q191, K193, E190, K194, G192, D138 Epi#29 G390,Q332, L329, R330, E435 V374, N366, L369, E348 I500, P495, L493, E496G344, Q332, L333, R330, E435 Epi#30 G412, N304, A306, H309, I312, P314,V419 I312, L311, A315, H309, P229, L136, P132 Epi#31 L329, Q332, N343,R330, F331, V386, D434 L333, Q332, N343, R330, F331, V386, D434 L58,Q501, N54, R59, F112, M459, F456, D205 L58, Q501, N54, R59, F112, M459,I454, D205 Epi#33 Q485, Y490, P494, S499, A497, R59 Q251, Y256, P292,S295, A296, R234 H153, F21, V16, S17, A182, K173 H153, F21, P18, S17,A182, K173 Epi#34 V431, P395, T432, G433, G412, T415, S414 V431, P388,T432, G412, G433, S414, T415 V419, P320, T321, G323, P322, Y416, S414V431, P395, T432, G390, G433, S414, T415 Epi#35 A371, L369, A362, S360,M359, I358 G372, L369, A362, S360, M359, I358 A365, L369, A362, S360,M359, I358 Epi#36 A362, A471, A476, V474, G361, S360, Q357, P350, A371,A365 A290, A288, A285, V253, Y256, S295, A296, W257 A288, A285, A287,V253, Y256, S295, A296, W257 Epi#37 P132, A135, L136, K194, N250 A135,A134, L136, K194, D138 P298, A301, L303, R234, N299 Epi#38 L356, G81,E348, A371, V374, L369, N366, P370, S351 L356, G81, E348, A371, V374,L369, N366, P370, S349 Epi#39 A411, E435, T432, P395, G393, L392 A1,E142, L35, R37, P34, G30, L27 A389, E435, T432, P395, G394, L392 Epi#40R330, L333, G390, T432, A411, S414 G393, L392, G394, T432, A411, S414R330, L333, G390, T432, A411, T415 Epi#41 P370, L369, V352, S351 P350,L369, V352, S351 Epi#42 L392, P395, S428, G430, P388, R330, Q332 Epi#44S360, Q363, D367, P370, Y347, A371, G372, T345 V253, Q191, D254, P292,W257, Y256, S295, A296, P298 S360, Q363, D367, P370, Y347, S349, V352,P350 V253, Q191, D254, P292, W257, Y256, S295, G214, P252 Epi#45 R409,P322, F418, Y416, N420, D313, V419 K423, P314, F418, Y416, N420, D313,V419 R175, P180, F21, Y176, R266, D166, G268 Epi#46 A296, R259, R234,P300, N299, A301 Y256, R259, R234, P300, N299, Q302 Epi#47 I212, S211,R234, L303, A301, N299, P300, P298 I212, S211, R234, V232, A301, N299,P300, P298 Epi#48 S158, Q160, P157, P155, V504 S499, Q501, P55, P155,V504 E488, Q485, P480, P479, V481 Epi#49 D367, L369, V352, P350, Q357,Q363, M359, N478 D367, L369, P370, P350, Q357, Q363, M359, N478 Epi#50D291, Y256, W257, S295, P298 D254, Y256, W257, T293, S295 Epi#51 D307,H309, E228, T218, P229, T231, H230 R234, H215, E216, T231, P229, H230,H309 D248, H215, E216, T231, P229, H230, H309 Epi#52 F69, A100, T98,R71, W75, T73, Q70 F97, A100, T98, R71, W75, T73, Q70 Natalase: Epi#01P344, P382, R387, R33, N32, S28, R31, T36 P344, P382, R387, R33, N29,S28, R31, T36 Epi#02 A87, N21, Q18, R24, S28, R31, R33 A87, K89, S83,R24, S28, R31, R33 Epi#03 L307, K305, H402, I404, Y398 L307, K305, H401,I404, Y398 L307, K305, A304, I404, Y398 Epi#04 R167, S166, Q168, R172,N171, I173 R177, Y131, S128, Q125, R123, N124, I127 Epi#05 G178, A180,N124, P120, G190, S187, H234, L195 G178, A180, N124, P120, G190, R123,S187, Y192 G178, A180, N124, P120, G190, S187, H234, Y192 Epi#06 A87,N21, D25, R24, Q18, P14 G145, N146, D150, T147, R144, P142 G143, N146,D150, T147, R144, P142 G450, N451, D447, T455, K452, P453 A87, N21, D25,R22, Q18, P14 G454, N451, D447, T455, K452, P453 A378, P382, D447, T455,K452, P453 Epi#07 G145, T147, D150, S149, R213, V208, P205, D201 Epi#08K305, D400, A304, H402, F399 K305, D400, A304, H401, F399 Epi#09 S79,S83, D25, R22, R24, H86, N90, S28, R31 N439, A460, N459, V444, K478,N417, T413, T414 Epi#10 E254, N249, R248, T245, F239, R212, R213 E254,N249, R248, T245, F239, R241, K275 Epi#11 F169, I173, Q170, D162 L195,I173, Q170, D162 Epi#12 Y192, E188 Y357, E354 Epi#13 H12, L13, P369,A375, P374, S372, P330, W11 H12, L13, P369, A375, P374, S372, P330, L334H12, L13, P369, A375, P374, S372, P330, G331 Epi#15 N451, P453, D447,I448, T449, A378 N451, P453, D447, I448, K452, G450 Epi#16 Q313, P316,Y357, R353, Q395, D397, D400, A304, N308 Q355, P316, Y357, G356, R353,D397, D400, A304, D302 Epi#17 A87, S83, R24, S28 A87, S28, R24, S83Epi#18 R33, N32, R31, S28, G92, N90 Epi#19 D16, N50, S48, Q49, R72, G69D25, N21, Q80, Q18, R24, A87 E82, T77, Q18, Q80, R72, G69 Epi#22 D461,A460, W463, W433 Epi#23 K478, N417, E410, N439, Q438, A460 K478, N417,E410, N439, Q438, A441 Epi#24 E332, G331, E335, P330, S372, A375, K379D381, K379, A375, P369, S372, P374, K377 Epi#25 R154, K138, W136, D162,N171 R213, R212, W217, E216, N249 R154, K138, W136, E134, N112 R241,K236, W183, D203, E206 Epi#26 W163, S166, E134, W136, V161, E117, E126W163, S166, E134, W136, V161, E117, D130 W163, S166, E134, W136, V161,E117, D162 Epi#27 D203, E206, D201, K236 E117, E126, D130, K175 D201,E206, D203, K179 E126, E117, D162, K175 Epi#28 L195, D162, Q168, W163,E134, W136, Q165, S166, R167 I173, D162, Q170, W163, E134, W136, Q165,S166, R167 V161, D162, Q170, W163, E134, W136, Q165, S166, R167 Epi#29G331, P330, L334, F337, E335 G178, K175, L114, R177, E117 Epi#30 G450,N451, H446, K478, I448, P453 G454, N451, H446, K478, I448, P453 Epi#31Q168, N171, R172, W163, M196, I173, D162 Q170, N171, R172, W163, V161,I173, D162 Epi#33 K377, Y366, P369, S372, A375, K379 K377, Y366, P374,S372, A375, K379 Epi#34 W433, W463, T457, V444, G454, T455, P453 W433,W463, T457, V456, G454, T455, P453 Epi#37 Y156, R177, L114, K175, D130T132, R177, L114, K175, N124 Epi#38 G429, E431, N469, P428, S472 G430,E431, N469, P428, S472 Epi#39 E10, H12, T370, P330, G331, L334 E10, L13,T370, P330, G331, L334 Epi#40 A378, A375, Y366, P369, S372 R177, L114,G178, Y156, K138, T110 A375, A378, Y366, P369, T370 Epi#41 P369, L13,V52, S48 Epi#42 P316, S281, G356, R353, Q355 P316, S281, G356, R353,Q395 Epi#44 V208, R213, W217, Y148, S149, G145, P142 S28, R33, D381,Y365, A378, A375, P369 L13, D16, P14, W11, Y362, A375, V373, T370 S333,D327, P330, W11, Y362, A375, V373, P369 Epi#45 D108, P142, F65, Y60,N146, D150, G145 D140, P142, F65, Y60, N146, D150, G145 Epi#46 Y392,R387, R33, P382, G450, G454 Y392, R387, R33, P382, Q388, G3 Epi#47 S83,S79, E82, I85, R24, A87, N90, R31, S28 A250, G252, E254, N249, R248,F256, N279, R241, S238 Epi#48 S372, H371, P374, P369, V373 Epi#49 D51,W11, L13, V52, P14, Q18, Q80, T77, N21 D51, W11, L13, V52, P14, Q18,Q80, T77, K74 Epi#50 D461, Y435, W433, W463, T457 D400, Y398, W433,W463, T457 D397, Y435, W433, W463, T457 Epi#51 T394, H396, D397, D400,K305, H402, H401 T455, H446, K478, T457, G442, Q438, W463 Epi#52 W136,A109, E134, R167, W163, N171, Q170 W136, A109, E134, R167, W163, N171,Q168 PD498: Epi#02 T262, K258, S260, F266, T198, Y196, R168, V166 T262,K258, S260, F266, T264, Y196, R168, V166 T141, N139, Q171, F170, S167,Y196, R168, V166 Epi#03 L99, K51, A49, I53, Y56 L99, K51, A49, I53, Y43Epi#04 R28, S331, Q333, K97, R50, 153 R28, S331, Q333, K97, R50, A49Epi#05 G108, A106, N107, G110, S109, S111, 159 G110, A106, N107, G108,S109, S111, L112 G108, A106, N107, G110, S111, S117, Y121 G108, A106,N107, G110, S111, S109, G135 G110, A106, L68, P214, G217, S219, Y220G108, A106, N107, G110, S111, S109, L134 Epi#06 G135, N163, D164, R168,S174, P176 G162, N165, D164, R168, S174, P176 A22, N274, D25, S2, S9, P6G154, N152, D148, T142, K144, P176 A22, P21, D25, S2, S9, P6 G154, N152,D148, S145, K144, P176 Epi#07 29, T332, S331, D95, S240, R28, V26, P21,D25 G29, T332, S330, D95, S331, R28, V26, P21, D25 Epi#08 K258, D257,S260, F266 K190, D185, S192, V207, F193 Epi#09 N215, N44, R50, 153, K54,N64, N63, R61 N44, A49, R50, 153, K54, N63, N64, R61 Epi#10 D188, N187,R189, S260, F266, G263, K258 D185, N187, R189, S260, F266, G263, K258Epi#12 Y268, E253 Epi#15 R50, P46, D82, I87, T83, G86 N215, P46, D82,I87, T83, G86 Epi#18 N216, N44, R50, 153, A49, P46, N215 N215, N44, R50,153, A49, P46, N216 Epi#19 D95, T332, S240, Q241, R28, G29 D95, T332,S330, Q241, R28, G29 Epi#22 D185, S192, D164, Y196, K267 D105, S111,D113, T141, K144 Epi#24 D95, K51, A49, P46, R50, K97 Epi#25 R120, K153,W151, D148, N152 R189, K190, D188, N187 R189, K190, D185, N208 Epi#27D201, E253, D257, K258 D257, E253, D201, K267 Epi#28 I259, D257, Q254,E253, K267, F266, S260, R189 I259, D257, Q254, E253, K267, F266, S260,K258 Epi#29 L68, G108, L134, F170, E137 G135, N163, L134, F170, E137Epi#30 G110, N107, A106, H71, L68, L104, L112 G108, N107, A106, H71,L68, P214, V213 G110, N107, A106, H71, P214, L68, L104 G110, N107, A106,H71, L68, L104, L134 Epi#33 Q12, Y220, V207, S222, S192, R189 190, F193,V207, S222, S192, R189 Q16, Y13, V207, S222, S192, R189 Epi#34 V26, W1,T27, G29, R28, S331, T332 W1, P21, T27, V26, R278, Y279, T255 Epi#35G135, L134, S225, M221, I209 G110, L134, S225, M221, I209 G108, L134,S225, M221, I209 G162, L134, S225, M221, I209 Epi#37 A49, V52, L99, K54,N63 SAS: 309, Size 17.16: Y121, A127, L99, K54, N63 SAS: 307, Size13.09: Y43, V52, L99, K54, N63 Epi#40 R189, G261, Y268, K258, S260 R189,G261, Y268, K258, T262 Epi#42 P3, S2, Q16, P21, R28, Q241 Epi#43 W199,Y196, G162, Q171, S140, L112, I115, T142 Epi#44 S145, D148, P176, W199,Y196, S167, G162, T169 S174, D201, P176, W199, Y196, S167, G197, T198Epi#47 S330, S331, R28, V26, A22, Q16, N17, P21, S2 G242, S240, R28,V26, A22, Q16, N17, P21, S2 G29, S331, R28, V26, A22, Q16, N17, P21, S2Epi#48 S2, D25, P21, P3, G86 S9, Q16, P21, P3, G86 Epi#50 R168, Y196,W199, T264, T198 D164, Y196, W199, T264, S260 Savinase: L21, N18, P14,R19, K231, N232, S236, Q239, S234 L21, N18, P14, R19, K231, N232, S234,Q230, S24 L21, N18, P14, R19, K231, N232, S234, Q230, T22 Epi#02 T254,N255, A188, R164, S158, Y186, R180, V197 T249, N263, Q12, R10, P14, R19,R269 T249, N263, S9, R10, P14, R19, R269

Epi#03

K27, A86, I43, Y89 Epi#04 K229, S234, Q230, K231, R269, A266 K27, S24,Q230, K231, R269, A15 K231, S234, Q239, R241, N246, A248 Epi#05 G187,A188, N255, T254, G252, S250, T249, L251 G189, A188, N255, T254, G252,S250, T249, L261 Epi#06 G252, N179, D175, S182, S154, P127 A188, N255,D191, R164, S158, P127 A188, N255, D191, R164, S128, P127 Epi#08 A131,E134, S139, A106, F49 A166, E134, S139, A106, F49 Epi#09 S103, T132,A131, E134, A166, R164, N167, S142, R143 Epi#10 D175, N177, N179, S182,F183, G155, R180 D175, N212, N153, S182, F183, G155, R180 Epi#11 F49,L94, 1105, Q107, V102, E134 F49, K92, I105, Q107, V102, E134 Epi#12Y161, E134 Y165, E134 Epi#13 S76, L73, P39, T207, A209, P204, S206,G205, Y208 S85, L73, P39, T207, A209, P204, S206, G205, Y203 Epi#16R164, P127, Y161, G152, S158, N255, D191, A166, N167 R164, P129, Y161,G152, S158, N255, D191, A166, N138 Epi#17 A156, S158, R164, S128 A188,S158, R164, S126 Epi#18 N177, N179, R180, S182, G155, S154, A156, S158N177, N178, R180, S182, G155, S154, N153, F183 Epi#19 D175, N179, S182,Q185, R180, L256 D175, N179, S182, Q185, R180, L251 I240, W235, S234,Q239, R241, K245 D175, N179, S182, Q185, R180, G252 Epi#23 R143, N114,E110, S139, Q135, A131 R143, N115, E110, N138, Q135, A131 Epi#24 D58,G59, E53, P51, F49, P54, Q57 D58, G59, E53, P51, S48, P54, Q57 D58, G59,E53, P54, S55, F49, Q107 Epi#25 R19, R269, E265, N18 R269, R19, E265,N18 Epi#28 V102, Q107, F49, E53, K92, Q57, G46, R44 A47, Q107, F49, E53,K92, Q57, G46, R44 V50, Q107, F49, E53, K92, Q57, G46, R44 Epi#29 I77,N74, L41, R44, E87 V4, N74, L41, R44, E87 G20, N18, L21, R19, E265Epi#30 G59, N60, S97, H62, L94, P51, P54 G98, N60, S97, H62, L94, P51,P54 Epi#31 L256, R180, N178, R10, W6, V197, D175 L251, R180, N178, R10,W6, V197, D175 Epi#33 Q107, F49, P51, S48, S55, K92 Q107, F49, P54, S55,A47, K92 Epi#34 V102, P129, S128, G125, R164, Y161, P127 V102, P129,S126, G125, R164, S158, P127

Epi#37

T254, A188, L256, R180, N177 T254, A188, L256, R180, N179 Epi#38 L94,G59, E53, A96, N60, P204, S206 L94, G59, E53, A96, N60, P204, S36 Epi#39A131, E134, L133, T132, P129, G125, L124 A166, E134, L133, T132, P129,G125, L124 Epi#40 R44, L41, G78, T207, P39, T37 R19, L21, G20, T22,K231, S234 R180, L256, G252, T254, A188, S158 Epi#41 P127, Y161, L133,V102, S99 P127, Y161, L133, V102, S103 P127, Y161, L133, V102, S101P127, Y161, L133, V102, S126 Epi#42 L73, P84, S85, N74, H17, P14, R19,R269 L80, P5, S3, N74, H17, P14, R19, R269 L21, P84, S85, N74, H17, P14,R19, R269 Epi#43 105, W111, A47, G46, Q57, S36, L41, I43, T37 Epi#44S126, R164, P127, Y161, S158, A188, T254 S128, R164, P129, Y161, S158,A188, T254 Epi#46 A15, R269, R19, P14, N18, G20 A266, R269, R19, P14,N18, A15 Epi#48 S55, Q57, P54, P51, G52 E53, Q57, P54, P51, G52 Epi#50R10, W6, S3, S76 R241, W235, S234, P233 R10, W6, V4, S9

Epi#51

Q239, H243, T247, R269, R19, K231, W235 R19, H17, E265, R269, K231,S234, W235 Epi#52 A15, S9, R10, W6, N198, Q176 A15, S9, R10, W6, N198,Q200 Amylase SP722: Epi#02 T419, N423, P422, F396, T5, Y398, R393, R37T419, N418, P422, F396, T5, Y398, R393, R37 Epi#03 L313, K311, H408,I410, Y404 L313, K311, H407, I410, Y404 Epi#04 R171, S170, Q172, R176,N175, I177 R181, Y135, S132, Q129, R127, N128, I131 Epi#05 G184, A186,N128, P124, G196, S193, H240, L201 G184, A186, N128, P124, G196, R127,S193, Y198 Epi#06 G147, N150, D154, T151, R148, P146 G149, N150, D154,T151, R148, P146 Epi#07 G149, T151, D154, S153, R219, V214, P211, D207Epi#08 K311, D406, A310, H407, F405 K311, D308, A310, H408, F405 Epi#09T461, R485, K484, N423, T419, N418 R485, K484, N423, T420, T419 Epi#10E260, N255, R254, T251, F245, R218, R219 T419, N423, N395, T5, F396,R393, R37 E260, T257, N255, T251, F245, R218, R219 Epi#11 F173, I177,Q174, D166 L201, I177, Q174, D166 Epi#12 Y363, E360 Y398, E360 Y198,E194 Epi#13 H16, L17, P375, A381, P380, S378, P336, W15 H16, L17, P375,A381, P380, S378, P336, G337 H16, L17, P375, A381, P380, S378, P336,L340 Epi#15 N457, P459, D453, I454, K458, G456 K458, P459, D453, I454,T455, A384 N457, P459, D453, I454, K458, G460 Epi#16 Q319, P322, Y363,R359, Q401, D403, D406, A310, N314 Q319, P322, Y363, G362, R359, D403,D406, A310, N314 Q319, P322, Y363, R359, R415, D403, D406, A310, N314Epi#17 A91, S32, R28, S87 A91, S87, R82, S83 Epi#18 R485, V450, G448,T463, T461, H452, V462 N126, N128, R127, G196, Y198, S193, N195, N125N25, R26, R28, S87, I89, A91, H90, N94 Epi#19 D20, N54, S52, Q53, R76,G73 D20, N19, Q22, Q84, R76, G73 D29, N25, Q22, Q84, R28, A91 Epi#20K385, P350, L355, L313, K311, D308, G305, D432 Epi#22 D183, A186, D209,W189, K242 D183, A186, D209, W189, E190 D183, A186, D209, P211, E212D209, A186, D183, Y160, W159 D183, A186, D209, W187, W189 Epi#23 R415,N418, E416, N445, Q444, A466 K446, N445, E416, Y441, Q444, A466 Epi#24D387, K385, A381, P375, S378, P380, K383 E341, G337, E338, P336, S378,A381, K385 D333, G337, E341, P336, S378, A381, K385 Epi#25 R485, H452,I454, E391, N36 R485, K484, I454, E391, N395 Epi#26 W167, S170, E138,W140, V117, G182, D183 W167, S170, E138, W140, V165, E121, D134 W167,S170, E138, W140, V165, E121, E130 Epi#27 E212, E216, D154, K156 E216,E212, D209, K242 Epi#28 L201, D166, Q172, W167, E138, W140, Q169, S170,R171 L201, D166, Q169, W140, E138, W167, F173, S170, R171 L201, D166,Q174, W167, E138, W140, Q169, S170, R171 Epi#29 V214, N215, L217, R219,E222 G96, H90, L228, R82, E86 V214, R219, L217, R218, E212 Epi#30 G456,N457, H452, K484, I454, P459 G362, M323, S287, H324, K320, P322, V318G362, M323, S287, H321, K320, P322, V318 G460, N457, H452, K484, I454,P459 Epi#31 L217, R219, N215, R218, F245, V214, D248 L217, R219, N215,R218, F245, M208, D209 Epi#33 K383, Y372, P375, S378, A381, K385 K383,Y372, P380, S378, A381, K38 Epi#34 W439, W469, T463, V450, R485, T461,P459 W439, W469, T463, V462, R485, T461, P459 Epi#37 T251, R218, L217,R219, N215 P211, V214, L217, R219, N215 A256, R218, L217, R219, N215Epi#38 G435, E437, N475, P434, S478 G436, E437, N475, P434, S478 Epi#39E338, H16, T376, P336, G337, L340 E14, H16, T376, P336, G337, L340Epi#40 A384, A381, Y372, P375, S378 A384, A381, Y372, P375, T376 Epi#41P375, L17, V56, S52 Epi#42 S378, P380, Y372, A381, A384, P375 S378,P375, Y372, A381, A384, P388 S378, P375, Y372, A381, A384, T455 Epi#45K72, P146, F69, Y64, R148, D154, G149 K311, H408, F405, N409, D432, G304D406, H408, F405, N409, D432, G304 Epi#46 Y398, R393, R37, P388, Q394,G7 Y398, R359, R393, P388, G456, G460 Y398, R393, R37, P388, Q394, G38Epi#47 A256, G258, E260, N255, R254, F262, N285, R247, S244 S193, Y198,E194, N125, R127, Q129, N123, R176, P124 Epi#48 S378, H377, P380, P375,V379 H16, H377, P375, P380, V379 Epi#49 D55, W15, L17, P18, Q22, Q84,T81, N25 D55, W15, L17, P18, Q22, Q84, T81, K78 Epi#50 D467, Y441, W439,W469, T463 D406, Y404, W439, W469, T463 D183, Y160, W159, W140, T114D403, Y441, W439, W469, T463 Epi#51 D406, H408, D308, K311, L313, Q319,H321 Epi#52 W140, A113, E138, R171, W167, N175, Q174 W140, A113, E138,R171, W167, D166, Q172 Amylase AA560: Epi#01 L390, P388, P350, K383,K385, N457, S478, R458, T461 L390, P388, P350, K383, K385, N457, S478,R458, T452 L390, P388, P350, K383, K385, N457, S478, R458, T455 Epi#02L390, K395, Q394, R393, T5, Y398, R359, R400 L173, K172, S170, T136,Y135, R118, R181 L173, R171, S170, T136, Y135, R118, R181 L390, K395,Q394, R393, T5, Y398, R400, R415 Epi#03 K438, H407, I410, Y404 Epi#04K172, S170, Q169, R171, N174, L173 R171, S170, Q169, K172, N175, I177Epi#05 G456, A459, R458, T461, G460, T452, T463, V450 G456, A459, R458,T452, G460, T461, T463, G448 Epi#06 A51, N54, D20, R76, Q71, P146 G73,A51, D55, S52, K72, P146 Epi#07 G456, T455, S384, D387, R393, P388, D453Epi#08 K259, S255, V222, H252, F245 K259, G258, A256, H252, F245 Epi#09N128, V131, R176, D166, K172, N175, N174, R171 Epi#10 467, N445, R444,F441, R415, R400 D467, A466, R444, F441, R415, R400 Epi#11 F69, K72,I75, Q53, V56, D55 Epi#12 Y16, E337 Y363, E360 Y198, E194 Epi#15 K385,P388, D453, I454, R458, A459 K385, P388, D387, I454, T452, A459 K385,P388, D387, I454, R458, G456 Epi#17 A87, S29, R28, S32 A91, S29, R28,S32 Epi#18 N445, R444, A466, T463, T461, N471, N437 N445, R444, A466,T463, T461, T452, V450 Epi#19 166, W167, S170, Q169, R171, K172 E138,W167, S170, Q169, R171, K172 E134, T136, S170, Q169, R171, K172 Epi#22D209, P211, D207, Y160, D183 Epi#23 R400, N418, E416, N445, Q449, A466R82, N83, E68, N70, F69, P146 Epi#24 E134, G133, E130, P124, R176, L173,K172 E134, K179, E130, P124, R176, L173, K172 Epi#25 R444, K446, W469,D467, N445 R171, K172, W167, D166, N175 R171, K172, W167, D166, N174Epi#26 W167, S170, E138, W140, V165, E121, E130 W167, S170, E138, W140,V165, E121, E134 W167, S170, E138, W140, V165, E121, D166 Epi#27 E130,E121, D166, K172 D36, E391, D387, K385 E134, E121, D166, K172 Epi#28L201, D166, Q169, W140, E138, K172, S170, R171 L173, D166, Q169, K172,E138, W167, S170, R171 Epi#29 V131, R176, L173, R171, E138 I177, N175,L173, R171, E138 I177, N174, L173, R171, E138 Epi#30 I39, N33, S29, H23,P18, L17, P375 G38, N33, S29, H23, L17, P375, P380 G362, M323, S287,H321, Q319, P322, V318 G417, N423, A420, H421, K395, L390, P388 G21,N25, S29, H23, P18, L17, P375 G399, N418, A420, H421, K395, L390, P388Epi#31 L173, R171, N174, R176, W167, M202, I177, D166 L173, R171, N174,R176, W167, V165, I177, D166 Epi#33 K108, Y58, V56, S52, A51, K72 Epi#34W439, W469, T463, V450, G460, T452, T461 W15, P18, T376, G378, P375,Y372, S384 W469, W439, S473, G460, R458, T461, T463 Epi#37 P124, R176,L173, K172, N175 P124, R176, L173, R171, N174 Epi#40 R400, G399, Y396,P422, T419 R400, G417, Y396, P422, T419

Epi#41

P375, Y16, L17, V56, S52 P18, Y16, L17, V56, S52 Epi#42 P350, S478,G433, H408, R310, Q311 P322, S287, N285, H324, R320, Q319 P322, S287,G362, H321, R320, Q319 Epi#44 L17, D20, P18, W15, Y368, A381, G378, T376L340, D333, P336, W15, Y368, A381, G378, P375 Epi#45 K72, P146, F69,Y64, N150, D144, G147 D112, P146, F69, Y64, N150, D144, G149 Epi#46Y398, R359, R393, P388, G456, A459 Y363, R359, R393, P388, Q394, G7Y363, R359, R393, P388, Q394, G38 Epi#47 I75, E68, R76, N83, R82, Q84,N90, R28, S29 G133, E134, E130, V131, R176, L173, N174, R171, S170Epi#48 S384, K383, P380, P375, G378 E337, H377, P380, P375, V379 Epi#50R444, W469, W439, S473, T461 D183, Y160, W159, W140, T114 Epi#51 R320,H321, Q319, P322, H324, H286 Epi#52 W140, A113, E138, R171, W167, D166,Q169 W140, A113, E115, R118, W159, T114, Q169 Protease A: Epi#01 L21,N18, P14, R19, K237, N238, S242, Q245, S240 L21, N18, P14, R19, K237,N238, S240, Q236, S24 Epi#02 T255, N269, Q12, R10, P14, R19, R275 T255,N269, S9, R10, P14, R19, R275 Epi#03 K27, A88, I44, Y91 Epi#04 K235,S240, Q236, K237, R275, A15 K27, S24, Q236, K237, R275, A15 K237, S240,Q245, R247, N252, A254 R145, S141, Q137, Y171, N173, A172 Epi#06 G61,N62, D60, T38, Q59, P55 G211, P210, D60, T38, Q59, P55 A98, N62, D60,T38, Q59, P55 G100, N62, D60, T38, Q59, P55 Epi#08 A131, E136, S141,A108, F50 A172, E136, S141, A108, F50 A98, E54, G53, V51, F50 Epi#09S162, S170, A172, N173, V244, H249, N252, S256, T260 S259, S256, T260,N261, L262, R186, N185, S188, N155 S162, S170, A172, N173, V244, H249,N248, N252, T255 S156, S162, N261, S259, L262, R186, N185, S188, N155Epi#10 D181, N183, N185, S188, F189, G157, R186 D181, N218, N155, S156,F189, G157, R186 Epi#12 Y171, E136 Y91, E89 Epi#13 S78, L75, P40, T213,A215, P210, S212, G211, Y209 S87, L75, P40, T213, A215, P210, S212,G211, Y214 Epi#16 L262, P194, Y192, G195, S162, N261, D197, A172, N140L262, P194, Y192, G157, S162, N261, D197, A172, N173 L262, P194, Y192,G161, S162, S170, D197, A172, N173 Epi#17 A138, S141, R145, S144 A108,S141, R145, S144 Epi#18 N185, N183, R186, L262, S259, T260, P194, N261N185, N183, R186, L262, Y192, T260, P194, S162 Epi#19 I246, W241, S240,Q245, R247, K251 D181, N185, S188, Q191, R186, L262 Epi#23 R145, N116,E112, S141, Q137, A138 R145, N117, E112, S141, Q137, A108 Epi#24 E136,G133, A131, P129, S103, F50, Q109 E136, G132, A131, P129, S103, A108,Q137 D60, G61, E54, P52, F50, P55, Q59 Epi#25 R275, R19, E271, N18Epi#28 G20, H17, Q12, E271, L21, Q236, S240, K237 A15, H17, Q12, E271,L21, Q236, S240, K237 Epi#29 V244, Q245, L148, R145, E112 V244, N173,L148, R145, E112 Epi#30 G61, N62, A98, H64, L96, P52, P55 G20, N18, A15,H17, S87, L75, P40 I79, N76, S87, H17, Q12, P14, V4 G100, N62, A98, H64,L96, P52, P55 Epi#31 L262, R186, N184, R10, W6, V203, D181 L257, R186,N184, R10, W6, V203, D181 Epi#33 Q109, F50, P52, S49, S56, K94 Q109,F50, P55, S56, A48, K94 Epi#34 W241, P239, S242, G146, R145, S141, S144I165, P194, T260, G258, R186, S188, S156 V104, P129, S130, G127, G102,S101, S99 V244, W241, S242, G146, R145, S141, S144 I165, P194, S170,G127, P129, S130, S103 Epi#37 P14, A15, L21, R19, N18 T143, R145, L148,R247, N252 T143, V244, L148, R145, N116 Epi#38 L96, G97, E54, A98, N62,P210, S212 L96, G97, E54, A98, N62, P210, S37 Epi#39 A15, E271, H17,R19, P14, G20, L21 A254, E271, H17, R19, P14, G20, L21 A272, E271, H17,R19, P14, G20, L21 Epi#40 R186, L257, G258, T260, P194, S162 R186, L262,G161, Y192, P194, T260 Epi#41 P194, Y192, L262, S259 P194, Y192, L196,S162 Epi#42 L82, P5, S3, N76, H17, P14, R19, R275 L82, P5, S9, Q12, H17,P14, R19, R275 Epi#43 W113, A48, G47, Q59, S37, L42, I44, T38 Epi#44V244, R247, D197, P194, Y192, S162, G195, T260 V244, R247, D197, P194,Y192, S170, G195, T260 S56, Q59, D60, P210, Y214, S212, G211, T38 S56,Q59, D60, P210, Y209, S212, G211, T38 Epi#46 A15, R275, R19, P14, N18,G20 A272, R275, R19, P14, N18, G20 A272, R275, R19, P14, N18, A15 Epi#47S130, A131, E136, N173, A172, N140, R145, S144 S105, A131, E136, N173,A172, N140, R145, S144 Epi#48 E54, Q59, P55, P52, G53 S56, Q59, P55,P52, G53 S49, Q59, P55, P52, G53 Epi#50 R10, W6, S3, S78 R10, W6, V4, S9R10, W6, V203, S188 Epi#51 Q245, H249, T253, R275, K237, S240, W241 R19,H17, E271, R275, K237, S240, W241 R145, H120, K27, S24, K237, S240, W241R145, H120, K235, K237, P239, S240, W241 Epi#52 A15, S9, R10, W6, N204,Q206 A15, S9, R10, W6, N204, Q182 Alcalase: Epi#01 L10, P5, P9, K15,K12, N269, S251, R249, T253 L82, P5, P9, K15, K12, N269, S251, R249,T253 Epi#02 T115, N141, A144, R145, S242, R247, R249 A138, N141, A144,R145, S242, R247, R249 Epi#03 L196, K170, A129, 1165, Y167 L196, K170,A194, I165, Y171 Epi#04 R145, Y143, S173, Q137, K136, T133, A134 K170,Y167, S132, Q137, K136, N141, A144 Epi#05 G53, A52, F50, G102, S105,S103, Y104 G53, A52, F50, G102, S101, S103, Y104 Epi#06 A24, N25, D120,R145, S242, P239 A144, N141, D140, R145, S242, P239 Epi#08 K265, E197,S260, A194, F261 A56, E54, G53, A52, F50 Epi#10 T162, N161, N163, A194,F261, G264, K265 E195, N161, N163, S158, F261, G258, K265 Epi#12 Y57,E54 Y262, E197 Epi#13 S38, A37, P40, T213, A215, H64, L217, G204, Y206S38, A37, P40, T213, A215, H64, S98, G100, G61 S87, L75, P40, T213,A215, H64, L217, G204, Y6 Epi#16 L10, P9, Y6, G204, S182, N183, D181,A187, N185 Q2, P5, Y206, G204, S182, N183, D181, A203, N218 L10, P9, Y6,G204, S182, N183, D181, A187, N155 Epi#17 A144, S244, R247, S252 A272,S252, R249, S244 A144, S244, R249, S251 A254, S252, R249, S244 Epi#18N141, R145, A144, Y143, S244, N248, S252 Epi#19 N248, S244, Q245, R249,A272 N240, S242, Q245, R249, A254 N240, S242, Q245, R249, L241 Epi#22D76, L82, D14, A18, K15 D181, L10, D14, A18, K15 Epi#23 K27, N117, E112,N141, Q137, A134 K27, N117, E112, N141, Q137, A138 K27, N117, E112,S109, F50, A52 Epi#24 D120, K27, A24, P86, F21, A18, K15 D14, K22, A24,P86, F21, A18, K15 D76, K22, A24, P86, S87, F21, K15 Epi#25 R249, R247,E197, E195 Epi#27 D172, E195, E197, K265 E197, E195, D172, K136 D172,E197, E195, K170 Epi#28 A18, D14, Q19, K15, E271, K12, Q17, S87, D76 V4,D14, Q17, K12, E271, K15, F21, A18, K22 Epi#29 L257, K265, L196, F261,E195 G53, N97, L96, F50, E54 Epi#30 G146, L241, S242, H238, K237, P239,L235 G146, L241, S236, H238, S242, P239, L235 Epi#33 K15, F21, P86, S87,A24, K27 K27, Y91, V45, S89, A24, K22 Epi#34 V4, P5, T3, G80, P40, S38,T211 V108, W113, T116, G118, R145, Y143, S244 V26, P239, S242, G146,R145, T115, T116 Epi#36 A52, A56, A48, V51, G102, Y104, S105, V108,A138, A134 A52, A56, A48, V51, G102, Y104, S103, V108, A134, A138 Epi#37Y262, A194, L196, K265, Y256 Y263, R186, L257, K265, Y256 Y256, A254,L257, K265, Y262 Epi#40 R186, L257, A254, Y256, K265, S252 R186, L257,G258, Y256, K265, S260 Epi#41 Y256, L257, S260 Y256, L257, S259 Epi#42L235, P239, S242, N248, R249, Q275 L241, P239, S242, Q245, R249, Q275Epi#44 S132, Q137, D140, Y143, A144, A138, T133 V108, Q137, D140, Y143,A144, A138, T133 S173, Q137, D140, Y143, A144, A138, T133 Epi#48 Q19,K15, P9, P5, V4 E271, K15, P9, P5, V4 Protease B: Epi#05 SAS: 454, Size24.86: G189, A188, R164, P127, G125, S99 SAS: 452, Size 15.92: G189,A188, R164, P127, G125, S128 SAS: 451, Size 24.86: G157, A188, R164,P127, G125, S99 SAS: 449, Size 15.92: G157, A188, R164, P127, G125, S128SAS: 445, Size 23.31: G189, A166, R164, P127, G125, S99 Epi#09 SAS: 446,Size 15.76: T254, G189, A166, R164, A188, S158 SAS: 312, Size 15.90:T22, G20, L21, R19, A15, S9 Epi#10 SAS: 460, Size 17.32: D175, N177,N179, S182, F183, G155, R180 SAS: 437, Size 16.70: D211, N212, N153,S182, F183, G155, R180 SAS: 424, Size 13.75: D175, N212, N153, S182,F183, G155, R180 SAS: 417, Size 16.70: D211, N212, N153, S154, F183,G155, R180 SAS: 404, Size 15.83: D175, N212, N153, S154, F183, G155,R180 Epi#12 SAS: 309, Size 13.46: P127, Y161, E134, P129 SAS: 292, Size9.37: R164, Y161, E134, P129 SAS: 287, Size 18.66: P127, Y161, E134,N138 SAS: 284, Size 16.85: P127, Y161, E134, N167 SAS: 275, Size 11.53:S128, Y161, E134, P129 Epi#17 SAS: 275, Size 15.84: A188, S158, R164,S126 SAS: 225, Size 12.79: A156, S158, R164, S126 Epi#18 SAS: 444, Size16.32: S250, K245, S259, L256, A188, T254, L251 SAS: 397, Size 14.14:S250, K245, S259, L256, G252, T254, L251 SAS: 397, Size 14.14: S250,K245, S259, L251, G252, T254, L256 SAS: 397, Size 14.14: S259, K245,S250, L251, G252, T254, L256 SAS: 396, Size 21.52: S158, R164, S126,V102, G100, S99, L124 Epi#19 SAS: 295, Size 15.06: D175, W6, S9, Q12,R10 SAS: 278, Size 21.23: E110, T141, S236, Q239, R241 Epi#23 SAS: 486,Size 19.88: R143, N114, E110, S139, Q135, A131 SAS: 473, Size 18.68:R19, N18, E265, L21, Q230, P233 SAS: 468, Size 15.74: R164, N167, E134,S139, Q135, A131 SAS: 463, Size 13.77: R164, N167, E134, S130, Q135,A131 SAS: 461, Size 21.98: R44, N42, E87, S24, Q230, P233 Epi#28 SAS:520, Size 19.27: V102, Q107, W111, E110, Q135, S139, R143 SAS: 492, Size24.70: V102, Q107, F49, E53, Q57, G46, R44 SAS: 480, Size 22.76: V50,Q107, W111, E110, Q135, S139, R143 SAS: 452, Size 19.08: V50, Q107, F49,E53, Q57, G46, R44 SAS: 441, Size 24.70: V102, Q107, E110, W111, F49,G46, R44 Epi#29 SAS: 239, Size 11.49: G20, N18, L21, E265 SAS: 224, Size11.49: G20, R19, L21, E265 SAS: 179, Size 16.62: 14, P14, L21, E265 SAS:175, Size 11.49: G20, K231, L21, E265 SAS: 153, Size 18.96: G25, Q230,L21, E265 Epi#30 SAS: 308, Size 24.27: G20, L21, A15, H17, S85, L73, P39Epi#31 SAS: 363, Size 21.72: L256, R180, N178, R10, W6, V197, D211 SAS:352, Size 22.95: L251, R180, N178, R10, W6, V197, D211 SAS: 350, Size21.62: L256, R180, N178, R10, W6, V197, D175 SAS: 339, Size 17.75: L251,R180, N178, R10, W6, V197, D175 Epi#34 SAS: 430, Size 18.33: V238, W235,S236, G144, R143, S139, S142 SAS: 430, Size 18.33: V238, W235, S236,G144, R143, S142, S139 SAS: 420, Size 13.98: V238, W235, S236, G144,R143, S142, T141 SAS: 420, Size 13.98: V238, W235, S236, G144, R143,T141, S142 SAS: 352, Size 18.33: V238, W235, S236, G144, R143, S139,T141 Epi#37 SAS: 415, Size 23.06: T254, A188, L256, R180, N177 SAS: 374,Size 18.08: T254, A188, L256, R180, N179 SAS: 335, Size 19.96: T254,A188, L256, R180, N178 Epi#39 SAS: 425, Size 16.00: A166, E134, R164,P127, G125, L124 SAS: 421, Size 16.36: A131, E134, R164, P127, G125,L124 SAS: 400, Size 16.00: A166, E134, R164, P129, G125, L124 SAS: 396,Size 16.36: A131, E134, R164, P129, G125, L124 SAS: 359, Size 16.00:A166, E134, T132, P129, G125, L124 Epi#40 SAS: 358, Size 15.76: A166,G189, Y186, A188, T254 SAS: 352, Size 15.76: A166, G189, T254, A188,S158 SAS: 326, Size 11.62: A96, G59, T56, P54, S55 SAS: 322, Size 15.30:G98, G59, T56, P54, S55 SAS: 318, Size 17.81: A188, G189, Y186, A156,S182 Epi#42 SAS: 528, Size 16.22: L21, P14, S9, Q12, H17, R19, R269Epi#44 SAS: 401, Size 15.10: L256, R180, Y186, S158, A188, T254 SAS:393, Size 15.52: L256, R180, Y186, A188, G189, T254 SAS: 390, Size18.46: L251, R180, Y186, S158, A188, T254 SAS: 382, Size 16.23: L251,R180, Y186, A188, G189, T254 SAS: 376, Size 22.23: V197, R180, Y186,S158, A188, T254 Epi#46 SAS: 559, Size 12.63: A15, R269, R19, P14, N18,G20 Epi#53 SAS: 298, Size 9.48: W235, S234, Q230, K231 SAS: 298, Size18.05: W235, S234, Q239, K245 SAS: 289, Size 9.48: W235, P233, Q230,K231 SAS: 283, Size 9.61: W235, S234, Q239, K229 SAS: 255, Size 14.51:W235, S236, Q239, K245 ProteaseC: Epi#05 SAS: 445, Size 23.34: G189,A166, R164, P127, G125, S99 SAS: 445, Size 24.90: G189, A188, R164,P127, G125, S99 SAS: 433, Size 24.90: G157, A188, R164, P127, G125, S99SAS: 427, Size 15.89: G189, A188, R164, P127, G125, S128 SAS: 427, Size15.50: G189, A166, R164, P127, G125, S128 Epi#09 SAS: 463, Size 15.74:T254, G189, A166, R164, A188, S158 SAS: 425, Size 15.74: D191, G189,A166, R164, A188, T254 SAS: 384, Size 13.57: D191, G189, A166, R164,A188, S158 Epi#10 SAS: 445, Size 17.28: D175, N177, N179, S182, F183,G155, R180 SAS: 431, Size 13.75: D175, N212, N153, S182, F183, G155,R180 SAS: 403, Size 15.83: D175, N212, N153, S154, F183, G155, R180 SAS:387, Size 16.14: D175, N178, N179, S182, F183, G155, R180 SAS: 373, Size16.76: D175, N212, N153, A156, F183, G155, R180 Epi#12 SAS: 292, Size13.45: P127, Y161, E134, P129 SAS: 287, Size 9.30: R44, Y89, E87, N42SAS: 284, Size 9.35: R164, Y161, E134, P129 SAS: 282, Size 9.35: R164,Y165, E134, P129 SAS: 272, Size 16.85: P127, Y161, E134, N167 Epi#16SAS: 547, Size 20.59: R164, P129, Y165, G189, S158, N255, D191, A166,N167 SAS: 543, Size 23.80: R164, P129, Y165, G189, S158, N255, D191,A166, N138 Epi#17 SAS: 267, Size 15.84: A188, S158, R164, S126 SAS: 231,Size 12.82: A156, S158, R164, S126 Epi#18 SAS: 449, Size 16.85: S182,R180, L256, A188, T254, L251 SAS: 426, Size 21.97: S126, R164, S158,A188, T254, L256 SAS: 407, Size 15.92: S182, R180, L251, G252, T254,L256 SAS: 407, Size 15.92: S182, R180, L256, G252, T254, L251 SAS: 391,Size 18.26: S182, R180, L256, G252, S250, L251 Epi#19 SAS: 293, Size15.04: D175, W6, S9, Q12, R10 SAS: 291, Size 17.13: D191, N242, S236,Q239, R241 SAS: 273, Size 21.24: E110, T141, S236, Q239, R241 Epi#23SAS: 463, Size 19.84: R143, N114, E110, S139, Q135, A131 SAS: 451, Size15.68: R164, N167, E134, S139, Q135, A131 SAS: 443, Size 21.95: R44,N42, E87, S24, Q230, P233 SAS: 440, Size 22.70: R143, N115, E110, S139,Q135, A131 SAS: 431, Size 15.11: R44, N42, E87, S85, L73, P39 Epi#28SAS: 402, Size 18.79: G59, Q57, E53, F49, G46, R44 SAS: 384, Size 20.81:A96, Q57, E53, F49, G46, R44 SAS: 376, Size 18.79: A47, Q57, E53, F49,G46, R44 Epi#31 SAS: 348, Size 21.63: L256, R180, N178, R10, W6, V197,D175 SAS: 342, Size 17.75: L251, R180, N178, R10, W6, V197, D175 Epi#33SAS: 399, Size 18.88: Q107, Y102, P129, S126, R164 SAS: 355, Size 15.95:Q135, Y165, P129, S126, R164 Epi#34 SAS: 424, Size 18.37: V238, W235,S236, G144, R143, S139, S142 SAS: 424, Size 18.37: V238, W235, S236,G144, R143, S142, S139 SAS: 408, Size 14.02: V238, W235, S236, G144,R143, S142, T141 SAS: 408, Size 14.02: V238, W235, S236, G144, R143,T141, S142 SAS: 346, Size 18.37: V238, W235, S236, G144, R143, T141,S139 Epi#37 SAS: 405, Size 23.05: T254, A188, L256, R180, N177 SAS: 364,Size 18.08: T254, A188, L256, R180, N179 SAS: 347, Size 19.96: T254,A188, L256, R180, N178 Epi#40 SAS: 368, Size 15.74: A166, G189, T254,A188, S158 SAS: 362, Size 15.74: A166, G189, Y186, A188, T254 SAS: 326,Size 17.80: A188, G189, Y186, A156, S182 SAS: 326, Size 23.72: A166,G189, Y186, A156, S182 SAS: 326, Size 17.80: G189, A188, Y186, A156,S182 Epi#41 SAS: 232, Size 19.49: P204, Y208, L211, V197, S210 Epi#44SAS: 445, Size 22.71: V238, R241, D191, Y186, S158, A188, T254 SAS: 429,Size 21.14: V238, R241, D191, Y186, A188, G189, T254 SAS: 410, Size22.71: V238, R241, D191, Y186, S158, G189, T254 SAS: 404, Size 23.33:V238, R241, D191, Y257, S250, G252, T254 SAS: 382, Size 23.33: V238,R241, D191, Y257, S253, G252, T254 Epi#46 SAS: 567, Size 12.67: A15,R269, R19, P14, N18, G20 Epi#53 SAS: 305, Size 9.43: W235, S234, Q230,K231 SAS: 303, Size 9.53: W235, S234, Q239, K229 SAS: 276, Size 9.43:W235, P233, Q230, K231 SAS: 259, Size 9.43: W235, S234, Q230, K229 SAS:233, Size 9.53: W235, S236, Q239, K229 ProteaseD: Epi#05 SAS: 453, Size24.94: G189, A188, R164, P127, G125, S99 SAS: 449, Size 23.37: G189,A166, R164, P127, G125, S99 SAS: 442, Size 24.94: G157, A188, R164,P127, G125, S99 SAS: 439, Size 15.91: G189, A188, R164, P127, G125, S128SAS: 435, Size 15.50: G189, A166, R164, P127, G125, S128 Epi#09 SAS:448, Size 15.77: T254, G189, A166, R164, A188, S158 Epi#10 SAS: 460,Size 17.32: D175, N177, N179, S182, F183, G155, R180 SAS: 428, Size13.76: D175, N212, N153, S182, F183, G155, R180 SAS: 403, Size 15.83:D175, N212, N153, S154, F183, G155, R180 SAS: 391, Size 16.15: D175,N178, N179, S182, F183, G155, R180 SAS: 372, Size 16.77: D175, N212,N153, A156, F183, G155, R180 Epi#12 SAS: 302, Size 13.47: P127, Y161,E134, P129 SAS: 290, Size 9.39: R164, Y161, E134, P129 SAS: 282, Size18.68: P127, Y161, E134, N138 SAS: 280, Size 16.87: P127, Y161, E134,N167 SAS: 270, Size 13.10: R164, Y161, E134, N138 Epi#17 SAS: 286, Size15.87: A188, S158, R164, S126 SAS: 250, Size 12.76: A156, S158, R164,S126 Epi#18 SAS: 446, Size 16.31: S250, K245, S259, L256, A188, T254,L251 SAS: 406, Size 14.13: S250, K245, S259, L256, G252, T254, L251 SAS:406, Size 14.13: S250, K245, S259, L251, G252, T254, L256 SAS: 406, Size14.13: S259, K245, S250, L251, G252, T254, L256 SAS: 388, Size 14.13:S250, K245, S259, L256, G252, T249, L251 Epi#19 SAS: 319, Size 15.07:D175, W6, S9, Q12, R10 SAS: 276, Size 21.28: E110, T141, S236, Q239,R241 Epi#23 SAS: 497, Size 19.86: R143, N114, E110, S139, Q135, A131SAS: 487, Size 15.77: R164, N167, E134, S139, Q135, A131 SAS: 478, Size13.78: R164, N167, E134, S130, Q135, A131 SAS: 477, Size 18.16: R143,N138, E134, S139, Q135, A131 SAS: 472, Size 22.70: R143, N115, E110,S139, Q135, A131 Epi#28 SAS: 554, Size 22.17: A101, Q107, I102, E134,Q135, S139, R143 SAS: 532, Size 19.36: 1102, Q107, W111, E110, Q135,S139, R143 SAS: 527, Size 22.79: V50, Q107, I102, E134, Q135, S139, R143SAS: 509, Size 24.76: 1102, Q107, F49, E53, Q57, G46, R44 SAS: 508, Size22.17: A101, Q107, W111, E110, Q135, S139, R143 Epi#31 SAS: 355, Size21.56: L256, R180, N178, R10, W6, V197, D175 SAS: 352, Size 17.71: L251,R180, N178, R10, W6, V197, D175 Epi#34 SAS: 457, Size 18.37: V238, W235,S236, G144, R143, S139, S142 SAS: 457, Size 18.37: V238, W235, S236,G144, R143, S142, S139 SAS: 447, Size 14.02: V238, W235, S236, G144,R143, S142, T141 SAS: 447, Size 14.02: V238, W235, S236, G144, R143,T141, S142 SAS: 374, Size 18.37: V238, W235, S236, G144, R143, T141,S139 Epi#37 SAS: 397, Size 23.08: T254, A188, L256, R180, N177 SAS: 361,Size 18.08: T254, A188, L256, R180, N179 SAS: 328, Size 19.98: T254,A188, L256, R180, N178 Epi#39 SAS: 425, Size 16.36: A131, E134, R164,P127, G125, L124 SAS: 423, Size 16.02: A166, E134, R164, P127, G125,L124 SAS: 399, Size 16.36: A131, E134, R164, P129, G125, L124 SAS: 397,Size 16.02: A166, E134, R164, P129, G125, L124 SAS: 379, Size 16.36:A131, E134, T132, P129, G125, L124 Epi#40 SAS: 354, Size 15.77: A166,G189, T254, A188, S158 SAS: 351, Size 15.77: A166, G189, Y186, A188,T254 SAS: 334, Size 17.81: G189, A188, Y186, A156, S182 SAS: 334, Size17.81: A188, G189, Y186, A156, S182 SAS: 330, Size 14.42: A166, G189,Y186, A188, S158 Epi#41 SAS: 217, Size 19.46: P204, Y208, L211, V197,S210 Epi#44 SAS: 407, Size 15.10: L256, R180, Y186, S158, A188, T254SAS: 404, Size 18.45: L251, R180, Y186, S158, A188, T254 SAS: 387, Size15.52: L256, R180, Y186, A188, G189, T254 SAS: 384, Size 16.23: L251,R180, Y186, A188, G189, T254 SAS: 373, Size 22.26: V197, R180, Y186,S158, A188, T254 Epi#46 SAS: 545, Size 12.69: A15, R269, R19, P14, N18,G20 Epi#53 SAS: 306, Size 18.06: W235, S234, Q239, K245 SAS: 277, Size9.52: W235, S234, Q239, K229 SAS: 276, Size 9.46: W235, S234, Q230, K231SAS: 268, Size 9.46: W235, P233, Q230, K231 SAS: 258, Size 14.50: W235,S236, Q239, K245 ProteaseE: Epi#05 SAS: 461, Size 15.49: G189, A166,R164, P127, G125, S128 SAS: 459, Size 15.90: G189, A188, R164, P127,G125, S128 SAS: 435, Size 15.49: G189, A166, R164, P127, G125, S126 SAS:433, Size 15.49: G189, A166, R164, P129, G125, S128 SAS: 433, Size15.86: G189, A188, R164, P127, G125, S126 Epi#06 SAS: 518, Size 14.10:G189, A188, D157, S158, R164, P127 SAS: 490, Size 15.98: G189, A188,D157, S158, R164, P129 SAS: 460, Size 14.60: G155, A156, D157, S158,R164, P127 SAS: 432, Size 17.71: G155, A156, D157, S158, R164, P129Epi#09 SAS: 482, Size 15.78: T254, G189, A166, R164, A188, S158 SAS:311, Size 15.91: T22, G20, L21, R19, A15, S9 Epi#10 SAS: 455, Size17.26: D175, N177, N179, S182, F183, G155, R180 SAS: 406, Size 13.76:D175, N212, N153, S182, F183, G155, R180 SAS: 383, Size 16.16: D175,N178, N179, S182, F183, G155, R180 SAS: 381, Size 15.82: D175, N212,N153, S154, F183, G155, R180 SAS: 347, Size 16.78: D175, N212, N153,A156, F183, G155, R180 Epi#12 SAS: 310, Size 13.48: P127, Y161, E134,P129 SAS: 306, Size 9.40: R164, Y161, E134, P129 SAS: 297, Size 9.40:R164, Y165, E134, P129 SAS: 285, Size 16.90: P127, Y161, E134, N167 SAS:281, Size 18.68: P127, Y161, E134, N138 Epi#16 SAS: 673, Size 19.67:R164, P127, Y161, G125, S126, S154, D157, A188, N255 SAS: 664, Size20.60: R164, P129, Y165, G189, S158, S154, D157, A188, N255 SAS: 645,Size 20.60: R164, P129, Y161, G125, S126, S154, D157, A188, N255 SAS:636, Size 14.89: R164, P127, Y161, G125, S126, S154, D157, A156, N153SAS: 627, Size 17.25: R164, P129, Y165, G189, S158, S154, D157, A156,N153 Epi#17 SAS: 305, Size 15.86: A188, S158, R164, S126 SAS: 270, Size12.73: A156, S158, R164, S126 Epi#18 SAS: 590, Size 17.32: S250, K246,S259, L256, A188, T254, L251 SAS: 551, Size 16.26: S259, K246, S250,L251, G252, T254, L256 SAS: 551, Size 16.26: S250, K246, S259, L251,G252, T254, L256 SAS: 551, Size 16.26: S250, K246, S259, L256, G252,T254, L251 SAS: 518, Size 16.26: S250, K246, S259, L251, G252, S253,L256 Epi#23 SAS: 471, Size 19.86: R143, N114, E110, S139, Q135, A131SAS: 467, Size 13.75: R164, N167, E134, S130, Q135, A131 SAS: 467, Size15.76: R164, N167, E134, S139, Q135, A131 SAS: 451, Size 22.69: R143,N115, E110, S139, Q135, A131 SAS: 446, Size 19.99: R143, N138, E134,S130, Q135, A131 Epi#28 SAS: 505, Size 19.43: 1102, Q107, W111, E110,Q135, S139, R143 SAS: 500, Size 22.22: A101, Q107, W111, E110, Q135,S139, R143 SAS: 499, Size 24.79: 1102, Q107, F49, E53, Q57, G46, R44SAS: 494, Size 24.56: A101, Q107, F49, E53, Q57, G46, R44 SAS: 441, Size24.79: 1102, Q107, E110, W111, F49, G46, R44 Epi#29 SAS: 216, Size 9.94:143, R44, L41, E87 SAS: 209, Size 10.85: L73, N42, L41, E87 SAS: 200,Size 13.98: G46, R44, L41, E87 SAS: 199, Size 11.98: G45, R44, L41, E87SAS: 197, Size 19.08: 177, N74, L41, E87 Epi#30 SAS: 318, Size 24.25:G20, L21, A15, H17, S85, L73, P39 SAS: 277, Size 24.25: G20, L21, A15,H17, S85, L41, P39 SAS: 258, Size 21.05: G20, L21, A15, H17, S85, L73,L41 Epi#31 SAS: 377, Size 21.62: L256, R180, N178, R10, W6, V197, D175SAS: 370, Size 17.72: L251, R180, N178, R10, W6, V197, D175 Epi#33 SAS:388, Size 15.92: Q135, Y165, P129, S126, R164 Epi#34 SAS: 420, Size18.35: V238, W235, S236, G144, R143, S139, S142 SAS: 411, Size 13.98:V238, W235, S236, G144, R143, S142, T141 SAS: 341, Size 18.35: V238,W235, S236, G144, R143, S139, T141 Epi#37 SAS: 412, Size 23.05: T254,A188, L256, R180, N177 SAS: 378, Size 18.07: T254, A188, L256, R180,N179 SAS: 340, Size 20.00: T254, A188, L256, R180, N178 Epi#39 SAS: 445,Size 16.04: A166, E134, R164, P127, G125, L124 SAS: 432, Size 16.40:A131, E134, R164, P127, G125, L124 SAS: 417, Size 16.04: A166, E134,R164, P129, G125, L124 SAS: 404, Size 16.40: A131, E134, R164, P129,G125, L124 SAS: 376, Size 16.04: A166, E134, T132, P129, G125, L124Epi#40 SAS: 374, Size 15.78: A166, G189, T254, A188, S158 SAS: 334, Size15.78: A166, G189, Y186, A188, T254 SAS: 317, Size 11.62: A96, G59, T56,P54, S55 SAS: 312, Size 15.30: G98, G59, T56, P54, S55 SAS: 307, Size15.49: G189, A166, Y165, P129, S128 Epi#41 SAS: 234, Size 19.50: P204,Y208, L211, V197, S210 SAS: 189, Size 19.50: P204, Y208, L211, V197,S215 Epi#42 SAS: 549, Size 16.42: L21, P14, S9, Q12, H17, R19, R269Epi#44 SAS: 398, Size 15.10: L256, R180, Y186, S158, A188, T254 SAS:391, Size 18.47: L251, R180, Y186, S158, A188, T254 SAS: 372, Size15.51: L256, R180, Y186, A188, G189, T254 SAS: 371, Size 12.26: L256,R180, Y257, S250, G252, T254 SAS: 367, Size 15.51: L256, R180, Y186,S158, G189, T254 Epi#46 SAS: 575, Size 12.75: A15, R269, R19, P14, N18,G20 Epi#47 SAS: 491, Size 19.28: G45, E87, I43, R44, L41, N42, P39, S206Epi#53 SAS: 202, Size 9.12: W235, P233, K231 SAS: 199, Size 9.12: W235,S234, K231 SAS: 182, Size 6.73: W235, P233, K229 SAS: 179, Size 7.76:W235, S234, K229 SAS: 131, Size 8.39: W235, S236, K229 Properase: Epi#05SAS: 456, Size 15.94: G189, A188, R164, P127, G125, S128 SAS: 453, Size15.52: G189, A166, R164, P127, G125, S128 SAS: 451, Size 15.94: G157,A188, R164, P127, G125, S128 SAS: 427, Size 15.94: G189, A188, R164,P129, G125, S128 SAS: 424, Size 15.52: G189, A166, R164, P129, G125,S128 Epi#09 SAS: 480, Size 15.73: T254, G189, A166, R164, A188, S158SAS: 302, Size 15.88: T22, G20, L21, R19, A15, S9 Epi#10 SAS: 470, Size17.27: D175, N177, N179, S182, F183, G155, R180 SAS: 446, Size 13.75:D175, N212, N153, S182, F183, G155, R180 SAS: 420, Size 15.84: D175,N212, N153, S154, F183, G155, R180 SAS: 396, Size 16.09: D175, N178,N179, S182, F183, G155, R180 SAS: 380, Size 16.78: D175, N212, N153,A156, F183, G155, R180 Epi#12 SAS: 296, Size 9.36: R164, Y161, E134,P129 SAS: 295, Size 13.45: P127, Y161, E134, P129 SAS: 291, Size 9.36:R164, Y165, E134, P129 SAS: 271, Size 14.70: R164, Y161, E134, N102 SAS:270, Size 13.45: P127, Y161, E134, N102 Epi#17 SAS: 283, Size 15.87:A188, S158, R164, S126 SAS: 241, Size 12.73: A156, S158, R164, S126Epi#18 SAS: 474, Size 16.26: S250, K245, S259, L256, A188, T254, L251SAS: 435, Size 14.14: S250, K245, S259, L256, G252, T254, L251 SAS: 398,Size 14.14: S259, K245, S250, L251, G252, S253, L256 Epi#19 SAS: 260,Size 21.26: E110, T141, S236, Q239, R241 Epi#23 SAS: 491, Size 19.86:R143, N114, E110, S139, Q135, A131 SAS: 482, Size 15.76: R164, N167,E134, S139, Q135, A131 SAS: 465, Size 22.69: R143, N115, E110, S139,Q135, A131 SAS: 462, Size 18.17: R143, N138, E134, S139, Q135, A131 SAS:439, Size 18.17: R143, N138, E110, S139, Q135, A131 Epi#28 SAS: 445,Size 22.79: V50, Q107, W111, E110, Q135, S139, R143 SAS: 426, Size19.06: V50, Q107, F49, E53, Q57, G46, R44 SAS: 370, Size 19.06: V50,Q107, E110, W111, F49, G46, R44 Epi#31 SAS: 347, Size 21.62: L256, R180,N178, R10, W6, V197, D175 SAS: 339, Size 17.74: L251, R180, N178, R10,W6, V197, D175 Epi#33 SAS: 368, Size 15.95: Q135, Y165, P129, S126, R164Epi#34 SAS: 445, Size 18.39: V238, W235, S236, G144, R143, S139, S142SAS: 436, Size 14.07: V238, W235, S236, G144, R143, S142, T141 SAS: 358,Size 18.39: V238, W235, S236, G144, R143, T141, S139 Epi#37 SAS: 415,Size 23.03: T254, A188, L256, R180, N177 SAS: 374, Size 18.04: T254,A188, L256, R180, N179 SAS: 341, Size 19.93: T254, A188, L256, R180,N178 Epi#39 SAS: 323, Size 11.55: A15, E265, H17, R19, P14, G20, L21SAS: 238, Size 12.13: A15, E265, H17, T22, P14, G20, L21 Epi#40 SAS:370, Size 15.73: A166, G189, T254, A188, S158 SAS: 360, Size 15.73:A166, G189, Y186, A188, T254 SAS: 324, Size 17.80: A188, G189, Y186,A156, S182 SAS: 321, Size 23.71: A166, G189, Y186, A156, S182 Epi#41SAS: 228, Size 19.53: P204, Y208, L211, V197, S210 Epi#42 SAS: 554, Size16.31: L21, P14, S9, Q12, H17, R19, R269 Epi#44 SAS: 406, Size 15.06:L256, R180, Y186, S158, A188, T254 SAS: 398, Size 18.38: L251, R180,Y186, S158, A188, T254 SAS: 395, Size 12.22: L256, R180, Y257, S250,G252, T254 SAS: 392, Size 15.49: L256, R180, Y186, A188, G189, T254 SAS:387, Size 12.22: L251, R180, Y257, S250, G252, T254 Epi#46 SAS: 581,Size 12.65: A15, R269, R19, P14, N18, G20 Epi#53 SAS: 297, Size 18.06:W235, S234, Q239, K245 SAS: 283, Size 9.54: W235, S234, Q239, K229 SAS:250, Size 9.46: W235, S234, Q230, K231 SAS: 249, Size 14.49: W235, S236,Q239, K245 SAS: 247, Size 9.46: W235, P233, Q230, K231 Release: Epi#05SAS: 461, Size 17.25: G158, A189, R165, P128, G126, S129 SAS: 439, Size17.22: G158, A189, R165, P128, G126, S127 SAS: 436, Size 17.25: G158,A189, S159, P128, G126, S129 SAS: 420, Size 17.25: G158, A189, R165,P130, G126, S129 SAS: 414, Size 17.22: G158, A189, S159, P128, G126,S127 Epi#09 SAS: 510, Size 22.37: T22, G20, R19, A15, R270, A267, T250SAS: 501, Size 22.37: L21, G20, R19, A15, R270, A267, T250 Epi#10 SAS:458, Size 17.50: D176, N178, N180, S183, F184, G156, R181 SAS: 424, Size13.68: D176, N213, N154, S183, F184, G156, R181 SAS: 407, Size 15.87:D176, N213, N154, S155, F184, G156, R181 SAS: 392, Size 16.18: D176,N179, N180, S183, F184, G156, R181 SAS: 362, Size 16.73: D176, N213,N154, A157, F184, G156, R181 Epi#12 SAS: 323, Size 9.38: R45, Y90, E88,N43 SAS: 312, Size 13.53: P128, Y162, E135, P130 SAS: 302, Size 9.46:R165, Y162, E135, P130 SAS: 296, Size 9.46: R165, Y166, E135, P130 SAS:295, Size 13.19: T255, Y187, E190, S159 Epi#18 SAS: 431, Size 15.20:S251, K246, S260, L257, A189, T255, L252 SAS: 398, Size 14.35: S251,K246, S260, L252, G253, T255, L257 SAS: 378, Size 14.35: S251, K246,S260, L257, G253, T250, L252 Epi#19 SAS: 285, Size 21.53: E111, T142,S237, Q240, R242 SAS: 275, Size 12.58: D119, T142, S237, Q240, R242Epi#23 SAS: 512, Size 22.29: R45, N43, E88, S24, Q231, P234 SAS: 476,Size 19.71: R144, N115, E111, S140, Q136, A132 SAS: 460, Size 13.83:R165, N168, E135, S131, Q136, A132 SAS: 455, Size 20.11: R144, N139,E135, S131, Q136, A132 SAS: 452, Size 15.83: R165, N168, E135, S140,Q136, A132 Epi#25 SAS: 293, Size 13.93: R45, K27, D119, E88 Epi#28 SAS:502, Size 19.99: V103, Q108, W112, E111, Q136, S140, R144 SAS: 476, Size21.74: V51, Q108, F50, E54, Q58, S37, R45 SAS: 472, Size 24.93: V103,Q108, F50, E54, Q58, G47, R45 SAS: 469, Size 23.18: V51, Q108, W112,E111, Q136, S140, R144 SAS: 439, Size 19.16: V51, Q108, F50, E54, Q58,G47, R45 Epi#31 SAS: 354, Size 21.73: L257, R181, N179, R10, W6, V198,D176 SAS: 348, Size 17.85: L252, R181, N179, R10, W6, V198, D176 Epi#33SAS: 396, Size 22.75: Q201, Y204, P205, S37, R45 SAS: 379, Size 22.75:Q201, Y209, P205, S37, R45 SAS: 357, Size 18.39: H63, Y204, P205, S37,R45 Epi#34 SAS: 466, Size 13.97: V239, W236, S237, G145, R144, S143,T142 SAS: 463, Size 18.37: V239, W236, S237, G145, R144, S140, S143 SAS:387, Size 18.37: V239, W236, S237, G145, R144, S140, T142 Epi#36 SAS:206, Size 22.37: T250, A267, A15, G20, T22 Epi#37 SAS: 400, Size 22.59:T255, A189, L257, R181, N178 SAS: 359, Size 17.59: T255, A189, L257,R181, N180 SAS: 334, Size 19.35: T255, A189, L257, R181, N179 Epi#39SAS: 464, Size 16.36: A167, E135, R165, P128, G126, L125 SAS: 444, Size16.52: A132, E135, R165, P128, G126, L125 SAS: 441, Size 16.36: A167,E190, R165, P128, G126, L125 SAS: 441, Size 18.98: A189, E190, R165,P128, G126, L125 SAS: 423, Size 16.36: A167, E135, R165, P130, G126,L125 Epi#40 SAS: 324, Size 11.66: A97, G60, T57, P55, S56 SAS: 316, Size17.09: G158, A189, Y187, A157, S183 SAS: 307, Size 14.92: G158, A157,Y187, A189, T255 SAS: 307, Size 15.34: G99, G60, T57, P55, S56 Epi#41SAS: 222, Size 19.74: P205, Y209, L212, V198, S211 Epi#42 SAS: 544, Size16.22: L21, P14, S9, Q12, H17, R19, R270 Epi#44 SAS: 421, Size 14.87:L257, R181, Y187, S159, A189, T255 SAS: 415, Size 18.81: L252, R181,Y187, S159, A189, T255 SAS: 389, Size 22.36: V198, R181, Y187, S159,A189, T255 SAS: 389, Size 21.81:144, R45, Y90, A48, V51, P52 SAS: 386,Size 19.16:144, R45, Y90, A48, V51, P55 Epi#46 SAS: 557, Size 14.54:A267, R270, R19, P14, N18, G20 SAS: 553, Size 12.63: A15, R270, R19,P14, N18, G20 SAS: 540, Size 13.10: A267, R270, R19, P14, N18, A15 SAS:444, Size 14.54: A267, R270, R19, P14, G20, A15 Epi#47 SAS: 627, Size16.22: A267, R270, A15, R19, L21, N18, P14, S9 SAS: 436, Size 15.11:A267, E266, A15, R19, L21, N18, P14, S9 Epi#51 SAS: 545, Size 21.66:L21, R19, H17, D75, S77, I78, S3, W6 SAS: 485, Size 21.66: L21, R19,H17, D75, Q2, I78, S3, W6 Epi#53 SAS: 328, Size 9.43: W236, S235, Q231,K232 SAS: 316, Size 9.43: W236, P234, Q231, K232 SAS: 301, Size 18.21:W236, S235, Q240, K246 SAS: 246, Size 14.68: W236, S237, Q240, K246

“SAS” is solvent accessible surface. “Size” is the total surface area ofthe epitope in A2.

Example 12

The object of this example is to provide evidence showing thatsubtilisins with an homology to BPN′ of as low as 44.8% reveal a similarepitope distribution as BPN′.

Alcalase, Protease B, Savinase, Esperase, and PD498 (which range from44.8% to 69.5% in sequence identity to BPN′) were epitope mapped asdescribed in the above example, and compared with epitope mapped BPN′(FIG. 1).

The data in FIG. 1 show a significant overlap between the areas on theprimary structure of the respective proteases. Overall, 6 regions wereidentified: 1-20, 35-65, 95-115, 130-145, 170-220, and 260-270.

Even better overlap between the epitope sequences can be found amongproteins of higher sequence identity, such as within the Savinase-likesubtilisins with more than 81% identity, preferably more than 85%, morepreferably more than 90%, even more preferably more than 96% or mostpreferably more than 98% identity.

Example 13 Wash Performance

The following example provides results from a number of washing teststhat were conducted under the conditions indicated

TABLE 9 Experimental conditions for evaluation of Subtilisin variantsI44V. Detergent OMO Acao Detergent dose 2.5 g/l PH 10.5 Wash time 14min. Temperature 25° C. Water hardness 9° dH Enzymes Subtilisin variantI44V Enzyme conc. 10 nM Test system 150 ml glass beakers with a stirringrod Textile/volume 5 textile pieces (Ø 2.5 cm) in 50 ml detergent Testmaterial EMPA117 from Center for Test materials, Holland

TABLE 10 Experimental conditions for evaluation of Subtilisin variantsQ12D. Detergent Persil Powder Detergent dose 4 g/l PH 10.5 Wash time 20min. Temperature 30° C. Water hardness 18° dH Enzymes Subtilisin variantQ12D Enzyme conc. 10 nM Test system 150 ml glass beakers with a stirringrod Textile/volume 5 textile pieces (Ø 2.5 cm) in 50 ml detergent Testmaterial EMPA116 from Center for Test materials, Holland

TABLE 11 Experimental conditions for evaluation of Subtilisin variantsQ12D. Detergent Tide Detergent dose 1 g/l PH 10.5 Wash time 10 min.Temperature 25° C. Water hardness 6° dH Enzymes Subtilisin variant Q12DEnzyme conc. 10 nM Test system 150 ml glass beakers with a stirring rodTextile/volume 5 textile pieces (Ø 2.5 cm) in 50 ml detergent Testmaterial EMPA117 from Center for Test materials, Holland

pH is adjusted to 10.5 which is within the normal range for a powderdetergent.

Water hardness was adjusted by adding CaCl₂ and MgCl₂ (Ca²⁺:Mg²⁺=2:1) todeionized water (see also Surfactants in Consumer Products—Theory,Technology and Application, Springer Verlag 1986). pH of the detergentsolution was adjusted to pH 10.5 by addition of HCl.

Measurement of reflectance (R) on the test material was done at 460 nmusing a Macbeth ColorEye 7000 photometer. The measurements were doneaccording to the manufacturers protocol.

The wash performance of the variants was evaluated by calculating aperformance factor:

P=(R _(Variant) −R _(Blank))/(R _(Savinase) −R _(Blank))

P: Performance factorR_(Variant): Reflectance of test material washed with variantR_(Savinase): Reflectance of test material washed with Savinase®R_(Blank): Reflectance of test material washed with no enzyme

The variants all have improved wash performance compared toSavinase®—i.e. P>1.

The variants can be divided into improvement classes designated withcapital letters:

Class A: 1<P≦1.5 Class B: 1.5<P≦2 Class C: P>2

TABLE 12 Subtilisin variants and improvement classes. Improvement classVariants C I44V, Q12D

As it can be seen from Table 12 SAVINASE® variants of the inventionexhibits an improvement in wash performance.

1-134. (canceled)
 135. A protease variant having modified immunogenicity as compared to a parent protease, obtainable by a method comprising the steps of: (a) obtaining antibody binding peptide sequences, (b) using the sequences to localise epitope sequences on the 3-dimensional structure of the parent protein, (c) defining an epitope area including amino acids situated within 5 Å from the epitope amino acids constituting the epitope sequence, (d) changing one or more of the amino acids defining the epitope area of the parent protein by genetic engineering mutations of a DNA sequence encoding the parent protein, (e) introducing the mutated DNA sequence into a suitable host, culturing said host and expressing the protein variant, and (f) evaluating the immunogenicity of the protein variant using the parent protein as reference.
 136. The protease variant of claim 135, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10: Position −6 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion; Position −5 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion; Position −4 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion; Position −2 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion; Position 3a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion; Position 28a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion; Position 44a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion; Position 44b to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion; Position 139 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion; Position 148 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion; Position 149 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion; Position 264a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion.
 137. The protease variant of claim 135, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10: Position −1 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, H; Position 1 to V, L, I, W, M, F, Y, S, T, R; Position 2 to G, V, I, M, F, N, Q, Y, S, T, H; Position 3 to W, M, F, N, Q, Y, S, D, E, R, H; Position 4 to V, L, W, M, F, Y, R; Position 5 to V, L, I, W, M, F, N, Q, Y, T, R, H; Position 6 to G, V, L, I, W, P, M, N, Q, T, D, E, R, H; Position 9 to G, V, L, I, W, P, M, F, Q, Y, S, T, R, H; Position 10 to G, A, V, I, W, P, M, N, Q, Y, S, T, D, E, R; Position 12 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E; Position 14 to V, L, I, W, P, M, F, N, Q, Y, T, R, H; Position 15 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, E, H; Position 17 to G, A, V, I, W, P, M, F, Y, H; Position 18 to G, A, L, I, W, P, M, F, N, Q, Y, T, D, E, H; Position 19 to A, V, I, W, M, F, N, Y, S, T, D, R, H; Position 20 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E; Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H; Position 22 to G, V, L, I, W, M, F, Y, S, T; Position 24 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R; Position 25 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H; Position 27 to G, L, I, W, P, M, F, Y, T, H; Position 38 to V, L, I, W, M, F, N, Q, Y, T, H; Position 39 to G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H; Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H; Position 42 to G, A, L, W, C, M, F, N, Q, Y, S, T, D, E, R, H; Position 43 to G, L, H; Position 44 to G, V, L, I, W, P, M, F, Y, S, T; Position 45 to G, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H; Position 46 to G, A, L, I, W, P, M, F, Y, H; Position 47 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H; Position 48 to A, L, I, P, M, F, N, Y, D, H; Position 49 to G, A, V, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H; Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H; Position 51 to V, L, I, W, M, F, N, Y, R; Position 52 to V, L, I, W, M, F, Y, S, T, R; Position 53 to A, V, L, I, W, M, F, N, Q, Y, S, D, E, H; Position 54 to V, L, I, W, M, F, S, R; Position 55 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H; Position 56 to G, V, L, I, W, M, F, N, Q, Y, S, T, H; Position 57 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 58 to L, W, M, F, N, Y, R; Position 59 to A, V, L, I, C, T, H; Position 61 to V, L, I, W, M, F, Y; Position 62 to G, A, L, W, M, F, N, Y, R; Position 64 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 75 to L; Position 79 to I; Position 80 to G; Position 87 to A, V, L, I, W, M, F, Q, Y, S, T, D, E, H; Position 89 to G, V, L, I, W, P, F, N, Y, T, E; Position 91 to G, A, V, L, I, W, P, M, N, Y, S, T, D, E, R, H; Position 98 to A; Position 99 to V, L, I, W, M, F, Q, Y, H; Position 100 to G, V, L, I, W, M, F, Y, R, H; Position 101 to V, I, W, M, F, N, Q, Y, H; Position 102 to V, L, I, W, M, F, Y, R, H, G; Position 108 to I; Position 109 to N, Position 112 to E; Position 113 to W; Position 115 to I; Position 117 to N; Position 118 to N; Position 126 to L; Position 127 to G, A, V, I, W, M, F, Y, R, H, L; Position 128 to I, W; Position 129 to W; Position 130 to W, F, Y, R; Position 131 to W, Y, R; Position 132 to L, W, M, F, Y, S, H; Position 133 to A, L, I, W, M, F, Y, R; Position 134 to L, I, W, F, N, Q, Y, R, H; Position 136 to G, A, W, P, N, Y, S, T, D, E, H; Position 137 to G, A, V, I, W, P, M, N, Y, H; Position 140 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, H; Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H; Position 143 to V, L, I, P, M, F, N, Y, R; Position 144 to L, W, P, M, F, N, Q, Y, S, D, E, R, H; Position 145 to G, V, L, I, W, M, F, Q, Y, D, E, R, H; Position 146 to G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H; Position 155 to V, L, I, W, M, F, Y, R; Position 156 to V, I, W, F, R; Position 157 to G, A, V, L, I, W, M, F, Y, T, R, H; Position 158 to V, L, I, W, M, F, Y; Position 159 to A, W, M, Y, T, R, H; Position 160 to W, M, F, Y, R, H; Position 161 to I, W, M, F, Y, H; Position 167 to R, K; Position 171 to D; Position 172 to G, A, V, L, I, S, T, H; Position 173 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, H; Position 181 to G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H; Position 182 to A, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, H; Position 183 to G, A, V, L, W, C, M, F, N, Q, Y, S, T, E, R, H; Position 184 to A, V, L, I, W, C, M, F, N, Q, Y, T, E, H; Position 185 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, E, H; Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H; Position 188 to G, A, V, L, W, F, S, R, K; Position 189 to W, F; Position 191 to A, V, L, I, W, M, F, Y, T, R, H; Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H; Position 194 to W, N, Q, Y, D, H; Position 195 to W, P, Y; Position 196 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H; Position 203 to V, F, Y, R, H; Position 204 to I, W, M, Y, H; Position 206 to F; Position 209 to Y, R; Position 210 to W, F, Y; Position 211 to L, W, M, F, Y, H; Position 212 to V, L, I, W, M, F, Y, T, R, H; Position 214 to W, Y, R; Position 215 to A, L, I, W, M, F, Y; Position 216 to A, L, I, W, M, F, Y, R; Position 217 to W, R; Position 218 to G, A, L, W, P, M, F, Y, R, H; Position 221 to S; Position 236 to S; Position 240 to N; Position 241 to W; Position 243 to N; Position 245 to Q; Position 247 to G, V, I, W, P, F, Y, S, T, R; Position 248 to W, P, F, Y, E, R, H; Position 249 to L, W, P, F, S, D, E, H; Position 251 to G, L, I, W, P, M, F, Y, H; Position 252 to G, A, W, P, N, Q, Y, T, E, R, H; Position 254 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, H; Position 255 to G, L, W, M, F, N, Y, T, D, H; Position 256 to G, A, V, L, I, W, M, F, Q, Y, S, T, D, H; Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H; Position 258 to G, A, V, L, I, W, C, M, F, N, Q, Y, S, T, E, K, H; Position 259 to A, V, I, W, M, F, N, Q, Y, S, T, E, R; Position 260 to L, I, W, M, F, Y, T, H; Position 261 to L, N, S, H; Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H; Position 263 to G, A, V, L, I, P, C, M, N, Q, Y, S, T, R, K; Position 265 to V, L, I, W, M, F, Y; Position 269 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, R, H; Position 271 to A, L, I, W, P, M, F, N, Y, S, T, R, H; Position 272 to G, A, V, L, I, W, P, M, F, N, Q, Y, T, D, E, H; Position 275 to G, A, V, L, I, W, M, F, N, Y, T, D.
 138. The protease variant of claim 135, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10: Position −1 to Deletion; Position 9 to Insertion, deletion; Position 10 to Insertion, deletion; Position 12 to Insertion, deletion; Position 14 to Insertion, deletion; Position 15 to Insertion, deletion; Position 17 to Insertion, deletion; Position 18 to Insertion, deletion; Position 19 to Insertion, deletion; Position 20 to Insertion, deletion; Position 21 to Insertion, deletion; Position 22 to Insertion, deletion; Position 24 to Insertion, deletion; Position 25 to Insertion, deletion; Position 46 to Insertion, deletion; Position 47 to Insertion, deletion; Position 48 to Insertion, deletion; Position 49 to Insertion, deletion; Position 50 to Insertion, deletion; Position 51 to Insertion, deletion; Position 52 to Insertion, deletion; Position 53 to Insertion, deletion; Position 54 to Insertion, deletion; Position 55 to Insertion, deletion; Position 58 to Insertion, deletion; Position 59 to Insertion, deletion; Position 61 to Insertion, deletion; Position 64 to Insertion, deletion; Position 78 to Insertion: Position 80 to Insertion; Position 91 to Insertion, deletion; Position 98 to Deletion; Position 99 to Deletion; Position 102 to Deletion; Position 105 to Insertion; Position 108 to Insertion; Position 109 to Insertion; Position 112 to Insertion; Position 113 to Insertion; Position 115 to Insertion; Position 116 to Insertion; Position 117 to Insertion; Position 118 to Insertion; Position 131 to Deletion; Position 134 to Insertion, deletion; Position 136 to Insertion, deletion; Position 137 to Insertion, deletion; Position 140 to Insertion, deletion; Position 141 to Insertion, deletion; Position 143 to Insertion, deletion; Position 144 to Insertion, deletion; Position 145 to Insertion, deletion; Position 146 to Insertion, deletion; Position 171 to Deletion; Position 172 to Deletion; Position 173 to Deletion; Position 181 to Deletion; Position 182 to Deletion; Position 183 to Deletion; Position 184 to Deletion; Position 185 to Deletion; Position 186 to Deletion; Position 188 to Deletion; Position 189 to Deletion; Position 191 to Deletion; Position 192 to Deletion; Position 195 to Deletion; Position 196 to Insertion, deletion; Position 221 to Insertion; Position 236 to Insertion; Position 237 to Insertion; Position 238 to Insertion; Position 239 to Insertion; Position 240 to Insertion; Position 241 to Insertion; Position 242 to Insertion; Position 243 to Insertion; Position 244 to Insertion; Position 245 to Insertion; Position 247 to Insertion, deletion; Position 248 to Insertion, deletion; Position 249 to Insertion, deletion; Position 251 to Insertion, deletion; Position 252 to Insertion, deletion; Position 254 to Insertion, deletion; Position 255 to Insertion, deletion; Position 256 to Insertion, deletion; Position 257 to Insertion, deletion; Position 258 to Insertion, deletion; Position 259 to Insertion, deletion; Position 260 to Insertion, deletion; Position 261 to Insertion, deletion; Position 262 to Insertion, deletion; Position 263 to Insertion, deletion; Position 265 to Insertion, deletion; Position 269 to Insertion, deletion; Position 271 to Insertion, deletion; Position 272 to Insertion, deletion; Position 275 to Insertion, deletion.
 139. The protease variant of claim 135, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10: Position 7 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H; Position 8 to G, A, L, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 13 to G, L, I, W, P, M, F, N, Q, Y, S, D, E, H; Position 16 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, D, E, R, H; Position 23 to G, A, V, L, I, W, M, F, Y, E, R, H; Position 26 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H; Position 28 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 29 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 33 to V, L, I, W, C, M, F, N, Q, Y, R, H; Position 35 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 36 to V, L, I, W, P, M, F, N, Y, S, T, R, H; Position 37 to L, I, W, M, F, N, Q, Y, S, R, H; Position 41 to G, V, L, I, W, M, F, N, Q, Y, S, T, R, H; Position 60 to G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H; Position 63 to G, A, V, L, I, W, M, F, Y, T, R, H; Position 73 to A; Position 74 to A; Position 81 to V; Position 82 to L; Position 86 to G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H; Position 88 to A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H; Position 92 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 93 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 94 to G, V, L, I, W, P, M, F, N, Y, T, D, E, K, H; Position 96 to L, W, F, Y, R, K; Position 97 to V, L, W, C, M, F, Y, H; Position 111 to I; Position 114 to A; Position 119 to M; Position 124 to M; Position 135 to G, L, P, C, N, Q, T, R, H; Position 138 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H; Position 142 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 147 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 151 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 162 to I, W, F, Y, R; Position 163 to V, W, M, F, H; Position 168 to G, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 169 to C, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y; Position 174 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 176 to G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 179 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 187 to A, V, L, I, W, M, F, Y, R; Position 190 to G, A, V, L, I, W, C, M, F, N, Q, Y, S, T, R, K, H; Position 193 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H; Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H; Position 198 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 205 to W, F, Y, R, K; Position 208 to A, V, L, I, W, C, M, F, Y, T, R, K, H; Position 219 to G, A, V, L, I, W, F, Y, R, H; Position 222 to M; Position 232 to A; Position 233 to L; Position 234 to I; Position 250 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H; Position 267 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H; Position 268 to G, V, L, I, W, C, M, N, Q, Y, S, T, D, E, R, K, H; Position 270 to G, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 273 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 274 to W, P, M, F, N, Q, Y, T, D, E, R, H.
 140. The protease variant of claim 135, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10: Position 13 to Insertion, deletion; Position 16 to Insertion, deletion; Position 23 to Insertion, deletion; Position 26 to Insertion, deletion; Position 28 to Insertion, deletion; Position 29 to Insertion, deletion; Position 35 to Deletion; Position 60 to Insertion, deletion; Position 63 to Insertion; Position 81 to Insertion; Position 82 to Insertion; Position 92 to Insertion, deletion; Position 93 to Insertion, deletion; Position 94 to Insertion, deletion; Position 96 to Deletion, Position 106 to Insertion, Position 111 to Insertion, Position 114 to Insertion, Position 119 to Insertion, Position 124 to Insertion, Position 138 to Insertion, deletion; Position 142 to Insertion, deletion; Position 147 to Insertion, deletion; Position 151 to Insertion, deletion; Position 174 to Insertion, deletion; Position 176 to Insertion, deletion; Position 179 to Insertion, deletion; Position 187 to Deletion; Position 190 to Deletion; Position 193 to Deletion; Position 197 to Insertion, deletion; Position 198 to Insertion, deletion; Position 232 to Insertion, Position 233 to Insertion, Position 234 to Insertion, Position 246 to Insertion, Position 250 to Insertion, deletion; Position 267 to Insertion, deletion; Position 268 to Insertion, deletion; Position 270 to Insertion, deletion; Position 273 to Insertion, deletion.
 141. The protease variant of claim 135, wherein the protease is a savinase-like subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10: Position 2 to G, V, I, M, F, N, Q, Y, S, T, H, Position 3 to W, M, F, N, Q, Y, S, D, E, R, H, Position 4 to V, L, W, M, F, Y, R, Position 6 to G, V, L, I, W, P, M, N, Q, T, D, E, R, H, Position 9 to G, V, L, I, W, P, M, F, Q, Y, S, T, R, H, insertion, deletion, Position 10 to G, A, V, I, W, P, M, N, Q, Y, S, T, D, E, R, insertion, deletion, Position 12 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, insertion, deletion, Position 14 to V, L, I, W, P, M, F, N, Q, Y, T, R, H, insertion, deletion, Position 15 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, E, H, insertion, deletion, Position 17 to G, A, V, I, W, P, M, F, Y, H, insertion, deletion, Position 18 to G, A, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion, deletion, Position 19 to A, V, I, W, M, F, N, Y, S, T, D, R, H, insertion, deletion, Position 20 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, insertion, deletion, Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 22 to G, V, L, I, W, M, F, Y, S, T, insertion, deletion, Position 24 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, insertion, deletion, Position 25 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 27 to G, L, I, W, P, M, F, Y, T, H, Position 37 to L, I, W, M, F, N, Q, Y, S, R, H, Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H, Position 42 to G, A, L, W, C, M, F, N, Q, Y, S, T, D, E, R, H, Position 43 to G, L, H, Position 44 to G, V, L, I, W, P, M, F, Y, S, T, Position 45 to G, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, Position 46 to G, A, L, I, W, P, M, F, Y, H, insertion, deletion, Position 47 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 48 to A, L, I, P, M, F, N, Y, D, H, insertion, deletion, Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion, Position 51 to V, L, I, W, M, F, N, Y, R, deletion, insertion, Position 54 to V, L, I, W, M, F, S, R, deletion, insertion, Position 55 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H, deletion, insertion, Position 57 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H, Position 58 to L, W, M, F, N, Y, R, insertion, deletion, Position 59 to A, V, L, I, C, T, H, insertion, deletion, Position 61 to V, L, I, W, M, F, Y, insertion, deletion, Position 64 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 75 to L, Position 78 to insertion, Position 79 to I, Position 87 to A, V, L, I, W, M, F, Q, Y, S, T, D, E, H, Position 89 to G, V, L, I, W, P, F, N, Y, T, E, Position 91 to G, A, V, L, I, W, P, M, N, Y, S, T, D, E, R, H, insertion, deletion, Position 98 to A, deletion, Position 100 to G, V, L, I, W, M, F, Y, R, H, Position 101 to V, I, W, M, F, N, Q, Y, H, Position 102 to V, L, I, W, M, F, Y, R, H, G, deletion, Position 109 to N, insertion, Position 112 to E, insertion, Position 113 to W, insertion, Position 116 to insertion, Position 117 to N, insertion, Position 126 to L, Position 127 to G, A, V, I, W, M, F, Y, R, H, L, Position 128 to I, W, Position 129 to W, Position 130 to W, F, Y, R, Position 131 to W, Y, R, deletion, Position 132 to L, W, M, F, Y, S, H, Position 133 to A, L, I, W, M, F, Y, R, Position 134 to L, I, W, F, N, Q, Y, R, H, insertion, deletion, Position 136 to G, A, W, P, N, Y, S, T, D, E, H, insertion, deletion, Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion, Position 140 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, H, insertion, deletion, Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion, Position 143 to V, L, I, P, M, F, N, Y, R, insertion, deletion, Position 144 to L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion, Position 145 to G, V, L, I, W, M, F, Q, Y, D, E, R, H, insertion, deletion, Position 146 to G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H, insertion, deletion, Position 155 to V, L, I, W, M, F, Y, R, Position 156 to V, I, W, F, R, Position 157 to G, A, V, L, I, W, M, F, Y, T, R, H, Position 158 to V, L, I, W, M, F, Y, Position 160 to W, M, F, Y, R, H, Position 161 to I, W, M, F, Y, H, Position 167 to R, K, Position 170 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; Position 171 to D, deletion, Position 172 to G, A, V, L, I, S, T, H, deletion, Position 173 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, H, deletion, Position 181 to G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H, deletion, Position 183 to G, A, V, L, W, C, M, F, N, Q, Y, S, T, E, R, H, deletion, Position 184 to A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion, Position 185 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion, Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion, Position 188 to G, A, V, L, W, F, S, R, K, deletion, Position 189 to W, F, deletion, Position 191 to A, V, L, I, W, M, F, Y, T, R, H, deletion, Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion, Position 194 to W, N, Q, Y, D, H, Position 195 to W, P, Y, deletion, Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion, Position 203 to V, F, Y, R, H, Position 206 to F, Position 209 to Y, R, Position 210 to W, F, Y, Position 212 to V, L, I, W, M, F, Y, T, R, H, Position 214 to W, Y, R, Position 216 to A, L, I, W, M, F, Y, R, Position 217 to W, R, Position 218 to G, A, L, W, P, M, F, Y, R, H, Position 221 to S, insertion, Position 236 to S, insertion, Position 237 to insertion, Position 239 to insertion, Position 240 to N, insertion, Position 241 to W, insertion, Position 242 to insertion, Position 244 to insertion, Position 245 to Q, insertion, Position 247 to G, V, I, W, P, F, Y, S, T, R, insertion, deletion, Position 248 to W, P, F, Y, E, R, H, insertion, deletion, Position 251 to G, L, I, W, P, M, F, Y, H, insertion, deletion, Position 252 to G, A, W, P, N, Q, Y, T, E, R, H, insertion, deletion, Position 255 to G, L, W, M, F, N, Y, T, D, H, insertion, deletion, Position 256 to G, A, V, L, I, W, M, F, Q, Y, S, T, D, H, insertion, deletion, Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion, deletion, Position 258 to G, A, V, L, I, W, C, M, F, N, Q, Y, S, T, E, K, H, insertion, deletion, Position 259 to A, V, I, W, M, F, N, Q, Y, S, T, E, R, insertion, deletion, Position 260 to L, I, W, M, F, Y, T, H, insertion, deletion, Position 261 to L, N, S, H, insertion, deletion, Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion, deletion, Position 263 to G, A, V, L, I, P, C, M, N, Q, Y, S, T, R, K, insertion, deletion, Position 265 to V, L, I, W, M, F, Y, insertion, deletion, Position 271 to A, L, I, W, P, M, F, N, Y, S, T, R, H, insertion, deletion, Position 272 to G, A, V, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion, deletion, Position 275 to G, A, V, L, I, W, M, F, N, Y, T, D, insertion, deletion.
 142. The protease variant of claim 141, wherein the savinase-like subtilisin comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10: Position 6 to G, V, L, I, W, P, M, N, Q, T, D, E, R, H, Position 9 to G, V, L, I, W, P, M, F, Q, Y, S, T, R, H, insertion, deletion, Position 10 to G, A, V, I, W, P, M, N, Q, Y, S, T, D, E, R, insertion, deletion, Position 14 to V, L, I, W, P, M, F, N, Q, Y, T, R, H, insertion, deletion, Position 15 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, E, H, insertion, deletion, Position 17 to G, A, V, I, W, P, M, F, Y, H, insertion, deletion, Position 18 to G, A, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion, deletion, Position 19 to A, V, I, W, M, F, N, Y, S, T, D, R, H, insertion, deletion, Position 20 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, insertion, deletion, Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 37 to L, I, W, M, F, N, Q, Y, S, R, H, Position 43 to G, L, H, Position 45 to G, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, Position 47 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion, Position 51 to V, L, I, W, M, F, N, Y, R, deletion, insertion, Position 54 to V, L, I, W, M, F, S, R, deletion, insertion, Position 59 to A, V, L, I, C, T, H, insertion, deletion, Position 89 to G, V, L, I, W, P, F, N, Y, T, E, Position 91 to G, A, V, L, I, W, P, M, N, Y, S, T, D, E, R, H, insertion, deletion, Position 101 to V, I, W, M, F, N, Q, Y, H, Position 109 to N, insertion, Position 112 to E, insertion, Position 113 to W, insertion, Position 127 to G, A, V, I, W, M, F, Y, R, H, L, Position 128 to I, W, Position 129 to W, Position 130 to W, F, Y, R, Position 131 to W, Y, R, deletion, Position 133 to A, L, I, W, M, F, Y, R, Position 136 to G, A, W, P, N, Y, S, T, D, E, H, insertion, deletion, Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion, Position 140 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, H, insertion, deletion, Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion, Position 143 to V, L, I, P, M, F, N, Y, R, insertion, deletion, Position 144 to L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion, Position 145 to G, V, L, I, W, M, F, Q, Y, D, E, R, H, insertion, deletion, Position 146 to G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H, insertion, deletion, Position 155 to V, L, I, W, M, F, Y, R, Position 157 to G, A, V, L, I, W, M, F, Y, T, R, H, Position 158 to V, L, I, W, M, F, Y, Position 160 to W, M, F, Y, R, H, Position 161 to I, W, M, F, Y, H, Position 167 to R, K, Position 170 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; Position 171 to D, deletion, Position 172 to G, A, V, L, I, S, T, H, deletion, Position 173 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, H, deletion, Position 181 to G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H, deletion, Position 184 to A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion, Position 185 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion, Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion, Position 188 to G, A, V, L, W, F, S, R, K, deletion, Position 189 to W, F, deletion, Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion, Position 194 to W, N, Q, Y, D, H, Position 195 to W, P, Y, deletion, Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion, Position 203 to V, F, Y, R, H, Position 210 to W, F, Y, Position 218 to G, A, L, W, P, M, F, Y, R, H, Position 236 to S, insertion, Position 237 to insertion, Position 239 to insertion, Position 240 to N, insertion, Position 241 to W, insertion, Position 242 to insertion, Position 244 to insertion, Position 245 to Q, insertion, Position 247 to G, V, I, W, P, F, Y, S, T, R, insertion, deletion, Position 251 to G, L, I, W, P, M, F, Y, H, insertion, deletion, Position 255 to G, L, W, M, F, N, Y, T, D, H, insertion, deletion, Position 256 to G, A, V, L, I, W, M, F, Q, Y, S, T, D, H, insertion, deletion, Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion, deletion, Position 258 to G, A, V, L, I, W, C, M, F, N, Q, Y, S, T, E, K, H, insertion, deletion, Position 260 to L, I, W, M, F, Y, T, H, insertion, deletion, Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion, deletion, Position 265 to V, L, I, W, M, F, Y, insertion, deletion, Position 271 to A, L, I, W, P, M, F, N, Y, S, T, R, H, insertion, deletion, Position 272 to G, A, V, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion, deletion, Position 275 to G, A, V, L, I, W, M, F, N, Y, T, D, insertion, deletion.
 143. The savinase-like subtilisin of claim 141, wherein the subtilisin has at least 81% homology to SEQ ID NO:
 24. 144. The savinase-like subtilisin of claim 141, wherein the subtilisin has any of the amino acid sequence of SEQ ID NO: 24, 26, 27, 28, 29, 30, 31, 32, 34,
 35. 145. The protein variant of claim 135, wherein the protease is a savinase-like subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10: Position 8 to G, A, L, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, Position 16 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion, Position 23 to G, A, V, L, I, W, M, F, Y, E, R, H, insertion, deletion, Position 26 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 35 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H, deletion, Position 38 to V, L, I, W, M, F, N, Q, Y, T, H, Position 39 to G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H, Position 41 to G, V, L, I, W, M, F, N, Q, Y, S, T, R, H, Position 60 to G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H, insertion, deletion, Position 73 to A, Position 74 to A, Position 80 to G, insertion, Position 81 to V, insertion, Position 86 to G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H, Position 88 to A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, Position 90 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion, Position 93 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 108 to I, insertion, Position 111 to I, insertion, Position 124 to M, insertion, Position 135 to G, L, P, C, N, Q, T, R, H, Position 142 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 147 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 148 to G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 149 to G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 151 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 163 to V, W, M, F, H, Position 168 to G, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, R, K, H, Position 169 to C, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y, Position 174 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 179 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 190 to G, A, V, L, I, W, C, M, F, N, Q, Y, S, T, R, K, H, deletion, Position 193 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion, Position 196 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 208 to A, V, L, I, W, C, M, F, Y, T, R, K, H, Position 213 to N, oN, E, Position 215 to A, L, I, W, M, F, Y, Position 232 to A, insertion, Position 233 to L, insertion, Position 234 to I, insertion, Position 246 to insertion, Position 250 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 254 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion, Position 267 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 268 to G, V, L, I, W, C, M, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 269 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, R, H, insertion, deletion, Position 273 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion.
 146. The savinase-like subtilisin of claim 145, wherein the subtilisin has at least 81% homology to SEQ ID NO:
 24. 147. The savinase-like subtilisin of claim 146, wherein the subtilisin has any of the amino acid sequence of SEQ ID NO: 24, 26, 27, 28, 29, 30, 31, 32, 34,
 35. 148. The protease variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 25 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 25: Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 27 to G, L, I, W, P, M, F, Y, T, H, Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion, Position 52 to V, L, I, W, M, F, Y, S, T, R, deletion, insertion, Position 55 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H, deletion, insertion, Position 129 to W, Position 133 to A, L, I, W, M, F, Y, R, Position 172 to G, A, V, L, I, S, T, H, deletion, Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion, Position 194 to W, N, Q, Y, D, H, Position 195 to W, P, Y, deletion, Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion, Position 242 to insertion, Position 249 to L, W, P, F, S, D, E, H, insertion, deletion, Position 252 to G, A, W, P, N, Q, Y, T, E, R, H, insertion, deletion, Position 254 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion, Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion, deletion, Position 260 to L, I, W, M, F, Y, T, H, insertion, deletion, Position 265 to V, L, I, W, M, F, Y, insertion, deletion, with the proviso that the amino acids of the parent enzyme are substituted to another amino acid.
 149. The protein variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 10 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10: Position 4 to V, L, W, M, F, Y, R, Position 38 to V, L, I, W, M, F, N, Q, Y, T, H, Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H, Position 43 to G, L, H, Position 47 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 49 to G, A, V, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 54 to V, L, I, W, M, F, S, R, deletion, insertion, Position 96 to L, W, F, Y, R, K, deletion, Position 99 to V, L, I, W, M, F, Q, Y, H, deletion, Position 113 to W, insertion, Position 131 to W, Y, R, deletion, Position 133 to A, L, I, W, M, F, Y, R, Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion, Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion, Position 144 to L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion, Position 170 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; Position 173 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, H, deletion, Position 181 to G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H, deletion, Position 185 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion, Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion, Position 188 to G, A, V, L, W, F, S, R, K, deletion, Position 194 to W, N, Q, Y, D, H, Position 203 to V, F, Y, R, H, Position 210 to W, F, Y, Position 211 to L, W, M, F, Y, H, Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion, deletion, Position 261 to L, N, S, H, insertion, deletion, Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion, deletion, Position 265 to V, L, I, W, M, F, Y, insertion, deletion. with the proviso that the amino acids of the parent enzyme are substituted to another amino acid.
 150. The protein variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 11 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 11: Position 38 to V, L, I, W, M, F, N, Q, Y, T, H, Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H, Position 45 to G, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, Position 47 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 49 to G, A, V, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion, Position 52 to V, L, I, W, M, F, Y, S, T, R, deletion, insertion, Position 53 to A, V, L, I, W, M, F, N, Q, Y, S, D, E, H, deletion, insertion, Position 56 to G, V, L, I, W, M, F, N, Q, Y, S, T, H, Position 58 to L, W, M, F, N, Y, R, insertion, deletion, Position 96 to L, W, F, Y, R, K, deletion, Position 97 to V, L, W, C, M, F, Y, H, Position 98 to A, deletion, Position 105 to insertion, Position 109 to N, insertion, Position 113 to W, insertion, Position 115 to I, insertion, Position 133 to A, L, I, W, M, F, Y, R, Position 136 to G, A, W, P, N, Y, S, T, D, E, H, insertion, deletion, Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion, Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion, Position 158 to V, L, I, W, M, F, Y, Position 159 to A, W, M, Y, T, R, H, Position 172 to G, A, V, L, I, S, T, H, deletion, Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion, Position 189 to W, F, deletion, Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion, Position 195 to W, P, Y, deletion, Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion, Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion, deletion, Position 261 to L, N, S, H, insertion, deletion, Position 265 to V, L, I, W, M, F, Y, insertion, deletion, with the proviso that the amino acids of the parent enzyme are substituted to another amino acid.
 151. The protein variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 33 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 33: Position −6 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y, insertion, deletion, Position −5 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y, insertion, deletion, Position −4 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, insertion, deletion, Position −2 to A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion, Position −1 to G, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, R, H, deletion, Position 1 to V, L, I, W, M, F, Y, S, T, R, Position 2 to G, V, I, M, F, N, Q, Y, S, T, H, Position 3a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion, Position 5 to V, L, I, W, M, F, N, Q, Y, T, R, H, Position 6 to G, V, L, I, W, P, M, N, Q, T, D, E, R, H, Position 7 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, Position 8 to G, A, L, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, Position 10 to G, A, V, I, W, P, M, N, Q, Y, S, T, D, E, R, insertion, deletion, Position 12 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, insertion, deletion, Position 13 to G, L, I, W, P, M, F, N, Q, Y, S, D, E, H, insertion, deletion, Position 14 to V, L, I, W, P, M, F, N, Q, Y, T, R, H, insertion, deletion, Position 15 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, E, H, insertion, deletion, Position 16 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion, Position 17 to G, A, V, I, W, P, M, F, Y, H, insertion, deletion, Position 18 to G, A, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion, deletion, Position 19 to A, V, I, W, M, F, N, Y, S, T, D, R, H, insertion, deletion, Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 22 to G, V, L, I, W, M, F, Y, S, T, insertion, deletion, Position 23 to G, A, V, L, I, W, M, F, Y, E, R, H, insertion, deletion, Position 24 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, insertion, deletion, Position 25 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 26 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 27 to G, L, I, W, P, M, F, Y, T, H, Position 28 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 28a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion, Position 29 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 33 to V, L, I, W, C, M, F, N, Q, Y, R, H, Position 35 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H, deletion, Position 37 to L, I, W, M, F, N, Q, Y, S, R, H, Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H, Position 42 to G, A, L, W, C, M, F, N, Q, Y, S, T, D, E, R, H, Position 43 to G, L, H, Position 44 to G, V, L, I, W, P, M, F, Y, S, T, Position 44a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion, Position 44b to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion, Position 46 to G, A, L, I, W, P, M, F, Y, H, insertion, deletion, Position 48 to A, L, I, P, M, F, N, Y, D, H, insertion, deletion, Position 51 to V, L, I, W, M, F, N, Y, R, deletion, insertion, Position 52 to V, L, I, W, M, F, Y, S, T, R, deletion, insertion, Position 53 to A, V, L, I, W, M, F, N, Q, Y, S, D, E, H, deletion, insertion, Position 55 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H, deletion, insertion, Position 56 to G, V, L, I, W, M, F, N, Q, Y, S, T, H, Position 57 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H, Position 58 to L, W, M, F, N, Y, R, insertion, deletion, Position 61 to V, L, I, W, M, F, Y, insertion, deletion, Position 64 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 75 to L, Position 81 to insertion, Position 86 to G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H, Position 87 to A, V, L, I, W, M, F, Q, Y, S, T, D, E, H, Position 88 to A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, Position 89 to G, V, L, I, W, P, F, N, Y, T, E, Position 91 to G, A, V, L, I, W, P, M, N, Y, S, T, D, E, R, H, insertion, deletion, Position 92 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 94 to G, V, L, I, W, P, M, F, N, Y, T, D, E, K, H, insertion, deletion, Position 96 to L, W, F, Y, R, K, deletion, Position 97 to V, L, W, C, M, F, Y, H, Position 98 to deletion, Position 101 to V, I, W, M, F, N, Q, Y, H, Position 102 to V, L, I, W, M, F, Y, R, H, G, deletion, Position 108 to I, insertion, Position 109 to N, insertion, Position 111 to insertion, Position 112 to E, insertion, Position 113 to W, insertion, Position 114 to insertion, Position 115 to I, insertion, Position 117 to N, insertion, Position 118 to N, insertion, Position 119 to M, insertion, Position 127 to G, A, V, I, W, M, F, Y, R, H, L, Position 133 to A, L, I, W, M, F, Y, R, Position 134 to L, I, W, F, N, Q, Y, R, H, insertion, deletion, Position 135 to G, L, P, C, N, Q, T, R, H, Position 136 to G, A, W, P, N, Y, S, T, D, E, H, insertion, deletion, Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion, Position 138 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 139 to G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion, Position 142 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 144 to L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion, Position 145 to G, V, L, I, W, M, F, Q, Y, D, E, R, H, insertion, deletion, Position 146 to G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H, insertion, deletion, Position 147 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 148 to G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 156 to V, I, W, F, R, Position 158 to V, L, I, W, M, F, Y, Position 160 to W, M, F, Y, R, H, Position 161 to I, W, M, F, Y, H, Position 162 to I, W, F, Y, R, Position 163 to V, W, M, F, H, Position 167 to R, K, Position 169 to C, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y, Position 170 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion, Position 171 to D, deletion, Position 174 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 176 to G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 182 to A, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, H, deletion, Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion, Position 188 to G, A, V, L, W, F, S, R, K, deletion, Position 191 to A, V, L, I, W, M, F, Y, T, R, H, deletion, Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion, Position 193 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion, Position 194 to W, N, Q, Y, D, H, Position 195 to W, P, Y, deletion, Position 196 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion, Position 198 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 203 to V, F, Y, R, H, Position 205 to W, F, Y, R, K, Position 215 to A, L, I, W, M, F, Y, Position 216 to A, L, I, W, M, F, Y, R, Position 217 to W, R, Position 219 to G, A, V, L, I, W, F, Y, R, H, Position 233 to insertion, Position 234 to I, insertion, Position 236 to insertion, Position 237 to insertion, Position 238 to insertion, Position 239 to insertion, Position 240 to insertion, Position 243 to insertion, Position 246 to insertion, Position 247 to G, V, I, W, P, F, Y, S, T, R, insertion, deletion, Position 249 to L, W, P, F, S, D, E, H, insertion, deletion, Position 252 to G, A, W, P, N, Q, Y, T, E, R, H, insertion, deletion, Position 254 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion, Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion, deletion, Position 264a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion, Position 270 to G, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 273 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 274 to W, P, M, F, N, Q, Y, T, D, E, R, H, Position 275 to G, A, V, L, I, W, M, F, N, Y, T, D, insertion, deletion, Position 276 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion, with the proviso that the amino acids of the parent enzyme are substituted to another amino acid.
 152. The protein variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 33 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 33: Position 5 to V, L, I, W, M, F, N, Q, Y, T, R, H, Position 22 to G, V, L, I, W, M, F, Y, S, T, insertion, deletion, Position 26 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 28 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 37 to L, I, W, M, F, N, Q, Y, S, R, H, Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H, Position 44 to G, V, L, I, W, P, M, F, Y, S, T, Position 51 to V, L, I, W, M, F, N, Y, R, deletion, insertion, Position 52 to V, L, I, W, M, F, Y, S, T, R, deletion, insertion, Position 55 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H, deletion, insertion, Position 58 to L, W, M, F, N, Y, R, insertion, deletion, Position 61 to V, L, I, W, M, F, Y, insertion, deletion, Position 64 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 87 to A, V, L, I, W, M, F, Q, Y, S, T, D, E, H, Position 97 to V, L, W, C, M, F, Y, H, Position 98 to deletion, Position 101 to V, I, W, M, F, N, Q, Y, H, Position 102 to V, L, I, W, M, F, Y, R, H, G, deletion, Position 109 to N, insertion, Position 112 to E, insertion, Position 118 to N, insertion, Position 127 to G, A, V, I, W, M, F, Y, R, H, L, Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion, Position 146 to G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H, insertion, deletion, Position 156 to V, I, W, F, R, Position 158 to V, L, I, W, M, F, Y, Position 161 to I, W, M, F, Y, H, Position 188 to G, A, V, L, W, F, S, R, K, deletion, Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion, Position 194 to W, N, Q, Y, D, H, Position 195 to W, P, Y, deletion, Position 203 to V, F, Y, R, H, Position 216 to A, L, I, W, M, F, Y, R, Position 236 to insertion, Position 237 to insertion, Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion, deletion, Position 264a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion, with the proviso that the amino acids of the parent enzyme are substituted to another amino acid.
 153. A subtilisin variant comprising one or more of the insertions, substitutions and/or deletions in any of the positions of claim
 136. 154. The variant of claim 153, wherein the subtilisin has at least 80% homology to SEQ ID NO;
 10. 155. A composition comprising the protein variant of claim
 135. 156. A DNA construct comprising a DNA sequence encoding a protein variant of claim
 135. 157. An expression vector comprising a DNA construct of claim
 156. 158. A host cell which is capable of expressing a polypeptide and comprising a DNA construct of claim
 156. 159. A host cell which is capable of expressing a polypeptide and which is transformed by an expression vector of claim
 157. 160. The host cell of claim 159, which is a fungal cell, an insect cell, a mammalian cell, or a plant cell.
 161. A method of producing a protein variant having reduced immunogenicity as compared to the parent protein, comprising: (a) culturing the host of claim 159 in a suitable culture medium to obtain expression and secretion of the protein into the medium, followed by (b) isolation of the protein from the culture medium. 